Novel genes related to glutaminyl cyclase

ABSTRACT

Novel glutaminyl-peptide cyclotransferase-like proteins (QPCTLs), which are isoenzymes of glutaminyl cyclase (QC, EC 2.3.2.5), and to isolated nucleic acids coding for these isoenzymes, all of which are useful for the discovery of new therapeutic agents, for measuring cyclase activity, and for determining the inhibitory activity of compounds against these glutaminyl cyclase isoenzymes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. Non-provisional applicationSer. No. 12/497,082, filed on Jul. 2, 2009, which is a Division of U.S.Non-provisional application Ser. No. 11/859,217, filed on Sep. 21, 2007,which claims benefit of U.S. Provisional Patent Application Ser. No.60/846,244, filed on Sep. 21, 2006, and U.S. Provisional PatentApplication Ser. No. 60/947,780, filed on Jul. 3, 2007. All of theseapplications are incorporated herein by reference in their entireties tothe extent permitted by law.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED IN COMPUTER READABLEFORM

The Sequence Listing, which is a part of the present disclosure,includes a computer readable form and a written sequence listingcomprising nucleotide and/or amino acid sequences of the presentinvention. The sequence listing information recorded in computerreadable form is identical to the written sequence listing. The subjectmatter of the Sequence Listing is incorporated herein by reference inits entirety.

FIELD OF THE INVENTION

The present invention relates to novel glutaminyl-peptidecyclotransferase-like proteins (QPCTLs), which are isoenzymes ofglutaminyl cyclase (QC, EC 2.3.2.5), and to isolated nucleic acidscoding for these isoenzymes, all of which are useful for the discoveryof new therapeutic agents, for measuring cyclase activity, and fordetermining the inhibitory activity of compounds against theseglutaminyl cyclase isoenzymes.

BACKGROUND OF THE INVENTION

Glutaminyl cyclase (QC, EC 2.3.2.5) catalyzes the intramolecularcyclization of N-terminal glutamine residues into pyroglutamic acid(pGlu*) liberating ammonia. A QC was first isolated by Messer from thelatex of the tropical plant Carica papaya in 1963 (Messer, M. 1963Nature 4874, 1299). 24 years later, a corresponding enzymatic activitywas discovered in animal pituitary (Busby, W. H. J. et al. 1987 J BiolChem 262, 8532-8536; Fischer, W. H. and Spiess, J. 1987 Proc Natl AcadSci USA 84, 3628-3632). For the mammalian QC, the conversion of Gln intopGlu by QC could be shown for the precursors of TRH and GnRH (Busby, W.H. J. et al. 1987 J Biol Chem 262, 8532-8536; Fischer, W. H. and Spiess,J. 1987 Proc Natl Acad Sci USA 84, 3628-3632). In addition, initiallocalization experiments of QC revealed a co-localization with itsputative products of catalysis in bovine pituitary, further improvingthe suggested function in peptide hormone synthesis (Bockers, T. M. etal. 1995 J Neuroendocrinol 7, 445-453). In contrast, the physiologicalfunction of the plant QC is less clear. In the case of the enzyme fromC. papaya, a role in the plant defense against pathogenic microorganismswas suggested (El Moussaoui, A. et al. 2001 Cell Mol Life Sci 58,556-570). Putative QCs from other plants were identified by sequencecomparisons recently (Dahl, S. W. et al. 2000 Protein Expr Purif 20,27-36). The physiological function of these enzymes, however, is stillambiguous.

The QCs known from plants and animals show a strict specificity forL-Glutamine in the N-terminal position of the substrates and theirkinetic behavior was found to obey the Michaelis-Menten equation (Pohl,T. et al. 1991 Proc Natl Acad Sci USA 88, 10059-10063; Consalvo, A. P.et al. 1988 Anal Biochem 175, 131-138; Gololobov, M. Y. et al. 1996 BiolChem Hoppe Seyler 377, 395-398). A comparison of the primary structuresof the QCs from C. papaya and that of the highly conserved QC frommammals, however, did not reveal any sequence homology (Dahl, S. W. etal. 2000 Protein Expr Purif 20, 27-36). Whereas the plant QCs appear tobelong to a new enzyme family (Dahl, S. W. et al. 2000 Protein ExprPurif 20, 27-36), the mammalian QCs were found to have a pronouncedsequence homology to bacterial aminopeptidases (Bateman, R. C. et al.2001 Biochemistry 40, 11246-11250), leading to the conclusion that theQCs from plants and animals have different evolutionary origins.

Recently, it was shown that recombinant human QC as well as QC-activityfrom brain extracts catalyze both, the N-terminal glutaminyl as well asglutamate cyclization. Most striking is the finding, thatcyclase-catalyzed Glu₁-conversion is favored around pH 6.0 whileGln₁-conversion to pGlu-derivatives occurs with a pH-optimum of around8.0.

Since the formation of pGlu-Aβ-related peptides can be suppressed byinhibition of recombinant human QC and QC-activity from pig pituitaryextracts, the enzyme QC is a target in drug development for treatment ofAlzheimer's disease.

EP 02 011 349.4 discloses polynucleotides encoding insect glutaminylcyclase, as well as polypeptides encoded thereby. This applicationfurther provides host cells comprising expression vectors comprisingpolynucleotides of the invention. Isolated polypeptides and host cellscomprising insect QC are useful in methods of screening for agents thatreduce glutaminyl cyclase activity. Such agents are useful aspesticides.

Inhibitors of QC, which also could be useful as inhibitors of QCisoenzymes, are described in WO 2004/098625, WO 2004/098591, WO2005/039548 and WO 2005/075436, which are incorporated herein in theirentirety, especially with regard to the structure of the inhibitors,their use and their production.

DEFINITIONS Enzyme Inhibitors

Reversible enzyme inhibitors: comprise competitive inhibitors,non-competitive reversible inhibitors, slow-binding or tight-bindinginhibitors, transition state analogs and multisubstrate analogs.

Competitive Inhibitors Show

-   -   i) non-covalent interactions with the enzyme,    -   ii) compete with substrate for the enzyme active site,

The principal mechanism of action of a reversible enzyme inhibitor andthe definition of the dissociation constant can be visualized asfollows:

The formation of the enzyme-inhibitor [E−I] complex prevents binding ofsubstrates, therefore the reaction cannot proceed to the normalphysiological product, P. A larger inhibitor concentration [I] leads tolarger [E−I], leaving less free enzyme to which the substrate can bind.

Non-Competitive Reversible Inhibitors

-   -   i) bind at a site other than active site (allosteric binding        site)    -   ii) cause a conformational change in the enzyme which decreases        or stops catalytic activity.

Slow-Binding or Tight-Binding Inhibitors

-   -   i) are competitive inhibitors where the equilibrium between        inhibitor and enzyme is reached slowly,    -   ii) (k_(on) is slow), possibly due to conformational changes        that must occur in the enzyme or inhibitor        -   a) are often transition state analogs        -   b) are effective at concentrations similar to the enzyme            conc. (subnanomolar K_(D) values)        -   c) due to k_(off) values being so low these types of            inhibitors are “almost” irreversible

Transition State Analogs

are competitive inhibitors which mimic the transition state of an enzymecatalyzed reaction. Enzyme catalysis occurs due to a lowering of theenergy of the transition state, therefore, transition state binding isfavored over substrate binding.

Multisubstrate Analogs

For a reaction involving two or more substrates, a competitive inhibitoror transition state analog can be designed which contains structuralcharacteristics resembling two or more of the substrates.

Irreversible enzyme inhibitors: drive the equilibrium between theunbound enzyme and inhibitor and enzyme inhibitor complex(E+I< - - - >E−I) all the way to the right with a covalent bond (˜100kcal/mole), making the inhibition irreversible.

Affinity Labeling Agents

-   -   Active-site directed irreversible inhibitors (competitive        irreversible inhibitor) are recognized by the enzyme        (reversible, specific binding) followed by covalent bond        formation, and        -   i) are structurally similar to substrate, transition state            or product allowing for specific interaction between drug            and target enzyme,        -   ii) contain reactive functional group (e.g. a nucleophile,            —COCH₂Br) allowing for covalent bond formation            -   The reaction scheme below describes an active-site                directed reagent with its target enzyme where K_(D) is                the dissociation constant and k_(inactivation) is the                rate of covalent bond formation.

-   -   Mechanism-based enzyme inactivators (also called suicide        inhibitors) are active-site directed reagents (unreactive) which        binds to the enzyme active site where it is transformed to a        reactive form (activated) by the enzyme's catalytic        capabilities. Once activated, a covalent bond between the        inhibitor and the enzyme is formed.    -   The reaction scheme below shows the mechanism of action of a        mechanism based enzyme inactivator, where K_(D) is the        dissociation complex, k₂ is the rate of activation of the        inhibitor once bound to the enzyme, k₃ is the rate of        dissociation of the activated inhibitor, P, from the enzyme        (product can still be reactive) from the enzyme and k₄ is the        rate of covalent bond formation between the activated inhibitor        and the enzyme.

-   -   Inactivation (covalent bond formation, k₄) must occur prior to        dissociation (k₃) otherwise the now reactive inhibitor is        released into the environment. Partition ratio, k₃/k₄: ratio of        released product to inactivation should be minimized for        efficient inactivation of the system and minimal undesirable        side reactions.    -   A large partition ratio (favors dissocation) leads to        nonspecific reactions.

Uncompetitive enzyme inhibitors: From the definition of uncompetitiveinhibitor (an inhibitor which binds only to ES complexes) the followingequilibria can be written:

The ES complex dissociates the substrate with a dissociation constantequal to Ks, whereas the ESI complex does not dissociate it (i.e has aKs value equal to zero). The K_(m)'s of Michaelis-Menten type enzymesare expected to be reduced. Increasing substrate concentration leads toincreasing ESI concentration (a complex incapable of progressing toreaction products), therefore the inhibition can not be removed.

Preferred according to the present invention are competitive enzymeinhibitors.

Most preferred are competitive reversible enzyme inhibitors.

The terms “k_(i)” or “K_(I)” and “K_(D)” are binding constants, whichdescribe the binding of an inhibitor to and the subsequent release froman enzyme. Another measure is the “IC₅₀” value, which reflects theinhibitor concentration, which at a given substrate concentrationresults in 50% enzyme activity.

The term “QC” as used herein comprises glutaminyl cyclase (QC), which issynonymous to glutaminyl-peptide cyclotransferase (QPCT); and QC-likeenzymes, which are synonymous to glutaminyl-peptidecyclotransferase-like proteins (QPCTLs). QC and QC-like enzymes haveidentical or similar enzymatic activity, further defined as QC activity.In this regard, QC-like enzymes can fundamentally differ in theirmolecular structure from QC.

“QC-activity” is defined as the catalytic activity of glutaminyl cyclase(QC, QPCT) and QC-like enzymes (QPCTLs). These enzymes are found invarious tissues of the body of a mammal including kidney, liver,intestine, brain and body fluids such as CSF, where they cyclizeglutamine or glutamate at the N-terminus of biologically active peptideswith a high specificity.

In particular, the term “QC activity” as used herein is defined asintramolecular cyclization of N-terminal glutamine residues intopyroglutamic acid (pGlu*) or of N-terminal L-homoglutamine orL-β-homoglutamine to a cyclic pyro-homoglutamine derivative underliberation of ammonia. See therefore schemes 1 and 2.

The term “EC” as used herein comprises the side activity of glutaminylcyclase (QC, QPCT) and QC-like enzymes (QPCTLs) as glutamate cyclase(EC), further defined as EC activity.

The term “EC activity” as used herein is defined as intramolecularcyclization of N-terminal glutamate residues into pyroglutamic acid(pGlu*) by glutaminyl cyclase (QC, QPCT) and QC-like enzymes (QPCTLs).See therefore scheme 3.

The term “QC-inhibitor” or “glutaminyl cyclase inhibitor” is generallyknown to a person skilled in the art and means enzyme inhibitors, whichinhibit the catalytic activity of glutaminyl cyclase (QC, QPCT) orQC-like enzymes (QPCTLs) or their glutamyl cyclase (EC) activity,preferably by direct interaction of the inhibitor with the enzyme.

The term “selective QC-inhibitor” as defined herein means enzymeinhibitors, which inhibit the catalytic activity of glutaminyl cyclase(QC, QPCT) but do not or with a lower potency inhibit at least oneQC-like enzymes (QPCTLs). Preferred are selective QC-inhibitors, whichinhibit glutaminyl cyclase (QC, QPCT) with an ki-value, which is oneorder of magnitude lower than its ki-value for the inhibition of atleast one QC-like enzyme (QPCTL). More preferably, the ki-value of saidselective QC-inhibitor for the inhibition of glutaminyl cyclase (QC,QPCT) is two orders of magnitude lower than its ki-value for theinhibition of at least one QC-like enzyme (QPCTL). Even more preferredare selective QC-inhibitors, wherein their ki-value for the inhibitionof glutaminyl cyclase (QC, QPCT) is three orders of magnitude lower thantheir ki-value for the inhibition of at least one QC-like enzyme(QPCTL). Most preferred are selective QC-inhibitors, which do notinhibit QC-like enzymes (QPCTLs).

The term “selective QPCTL-inhibitor” as defined herein means enzymeinhibitors, which inhibit the catalytic activity of at least one QC-likeenzyme (QPCTL), but do not or with a lower potency inhibit the activityof glutaminyl cyclase (QC, QPCT). Preferred are selectiveQPCTL-inhibitors, which inhibit at least one QC-like enzyme (QPCTL) withan ki-value, which is one order of magnitude lower than its ki-value forthe inhibition of glutaminyl cyclase (QC, QPCT). More preferably, theki-value of said selective QPCTL-inhibitor for the inhibition of atleast one QC-like enzyme (QPCTL) is two orders of magnitude lower thanits ki-value for the inhibition of glutaminyl cyclase (QC, QPCT). Evenmore preferred are selective QPCTL-inhibitors, wherein their ki-valuefor the inhibition of at least one QC-like enzyme (QPCTL) is threeorders of magnitude lower than their ki-value for the inhibition ofglutaminyl cyclase (QC, QPCT). Most preferred are selectiveQPCTL-inhibitors, which do not inhibit the activity of glutaminylcyclase (QC, QPCT).

Potency of QC Inhibition

In light of the correlation with QC inhibition, in preferredembodiments, the subject method and medical use utilize an agent with aK_(i) for QC inhibition of 10 μM or less, more preferably of 1 μM orless, even more preferably of 0.1 μM or less or 0.01 μM or less, or mostpreferably 0.01 μM or less. Indeed, inhibitors with K_(i) values in thelower micromolar, preferably the nanomolar and even more preferably thepicomolar range are contemplated. Thus, while the active agents aredescribed herein, for convenience, as “QC inhibitors”, it will beunderstood that such nomenclature is not intending to limit the subjectof the invention to a particular mechanism of action.

Molecular Weight of QC Inhibitors

In general, the QC inhibitors of the subject method or medical use willbe small molecules, e.g., with molecular weights of 1000 g/mole or less,500 g/mole or less, preferably of 400 g/mole or less, and even morepreferably of 350 g/mole or less and even of 300 g/mole or less.

The term “subject” as used herein, refers to an animal, preferably amammal, most preferably a human, who has been the object of treatment,observation or experiment.

The term “therapeutically effective amount” as used herein, means thatamount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue system, animal or humanbeing sought by a researcher, veterinarian, medical doctor or otherclinician, which includes alleviation of the symptoms of the disease ordisorder being treated.

As used herein, the term “pharmaceutically acceptable” embraces bothhuman and veterinary use: for example the term “pharmaceuticallyacceptable” embraces a veterinarily acceptable compound or a compoundacceptable in human medicine and health care.

Guillain-Barré Syndrome (GBS)

Alternative names are Landry-Guillain-Barré syndrome, Acute idiopathicpolyneuritis, Infectious polyneuritis or Acute inflammatorypolyneuropathy.

Guillain-Barré syndrome is a serious disorder that occurs when thebody's defense (immune) system mistakenly attacks part of the nervoussystem. This leads to nerve inflammation that causes muscle weakness,which continues to get worse.

Guillain-Barré syndrome is an autoimmune disorder. The exact cause ofGuillain-Barré syndrome is unknown. The syndrome may occur at any age,but is most common in people of both sexes between the ages 30 and 50.It often follows a minor infection, usually a respiratory (lung)infection or gastrointestinal (gut) infection. Usually, signs of theoriginal infection have disappeared before the symptoms ofGuillain-Barré begin. Guillain-Barré syndrome causes inflammation thatdamages parts of nerves. This nerve damage causes tingling, muscleweakness, and paralysis. The inflammation usually affects the nerve'scovering (myelin sheath). Such damage is called demyelination.Demyelination slows nerve signaling. Damage to other parts of the nervecan cause the nerve to stop working.

Symptoms of Guillain-Barré get worse very quickly. It may take only afew hours to reach the most severe symptoms. Muscle weakness or the lossof muscle function (paralysis) affects both sides of the body. If themuscle weakness starts in the legs and then spreads to the arms, it iscalled ascending paralysis.

Patients may notice tingling, foot or hand pain, and clumsiness. As theloss of muscle function gets worse, the patient may need breathingassistance.

There is no cure for Guillain-Barré syndrome. However, many treatmentsare available to help reduce symptoms, treat complications, and speed uprecovery. When symptoms are severe, the patient will need to go to thehospital for breathing help, treatment, and physical therapy. A methodcalled plasmaphoresis is used to remove a person's blood and replace itwith intravenous fluids or donated blood that is free of antibodies.High-dose immunoglobulin therapy is another procedure used to reduce theseverity and length of Guillain-Barré symptoms. Other treatments aredirected at preventing complications.

Chronic Inflammatory Demyelinizing Polyradiculoneuropathy (CIDP)

A disease, which resembles GBS but is characterized by a chronic courseis called chronic inflammatory demyelinizing polyradiculoneuropathy(CIDP). There is as yet no generally applicable definition for CIDP withthe exception of the observation that in contrast to GBS, theprogressive phase lasts longer than four weeks, often longer than sixmonths, and that deficiencies often remain in the patient. Themechanism, which causes the severe paresis with GBS and CIDP possiblyincludes an immune reaction and inflammation mediated by T lymphocytes,which follows demyelinization of peripheral neurons. This assumption isconfirmed by increased amounts of complement compounds and cytokinesobserved in the serum and cerebrospinal fluid of GBS patients. Theprocess of demyelinization, especially in the region of the nerve roots,is currently regarded as the decisive mechanism in the development ofnerve conduction block. One theory is based on a disorder of theblood/cerebrospinal fluid (CSF) barrier as a relatively early importantstep in the development of the disease. Another theory claims that leaksdevelop in the blood/CSF barrier as a consequence of the disease andcause the increased protein content in the CSF. At any rate,non-specific serum constituents without direct reference to the immunesystem could penetrate into the CSF from the blood, cause neuronal orglial dysfunctions and/or modify neuronal activity. An alternativemechanism is a reduced flow rate of the CSF, which could explain theincreased protein content of the CSF. This interpretation requires noimpairment or modified selectivity of the blood/CSF barrier. Althoughall the effects mentioned could be of importance for the course of GBSand CIDP, their actual contribution to the symptoms has not yet beenclarified. It has not been possible to establish a connection betweenthe increased protein concentrations in the CSF and specificelectrophysiological findings or the clinical picture. Factors in theCSF of GBS patients and multiple sclerosis patients, which interact withpotential-dependent sodium channels have recently been described (Wüz etal. 1995, Muscle and Nerve 18, 772-781). Brinkmeier (Brinkmeier et al.1996, Muscle and Nerve 19, 54-62) report that the factors have amolecular weight of less than three kDa, and under more stringent testconditions of less than one kDa. On the basis of this observation andthe fact that the activity of the factors was not substantially reducedeven after incubation of CSF with proteases, the authors concluded thatthe factors were neither antibodies nor cytokines.

Multiple Sclerosis (MS)

Multiple sclerosis is an autoimmune disease that affects the centralnervous system (the brain and spinal cord). Multiple sclerosis usuallyaffects woman more than men. The disorder most commonly begins betweenages 20 and 40, but can strike at any age. The exact cause is not known,but MS is believed to result from damage to the myelin sheath, theprotective material, which surrounds nerve cells. It is a progressivedisease, meaning the damage gets worse over time. Inflammation destroysthe myelin, leaving multiple areas of scar tissue (sclerosis). Theinflammation occurs when the body's own immune cells attack the nervoussystem. The inflammation causes nerve impulses to slow down or becomeblocked, leading to the symptoms of MS. Repeated episodes, or flare ups,of inflammation can occur along any area of the brain and spinal cord.Symptoms vary because the location and extent of each attack varies.Usually episodes that last days, weeks, or months alternate with timesof reduced or no symptoms (remission). Recurrence (relapse) is commonalthough non-stop progression without periods of remission may alsooccur.

It is not clear what triggers an attack. Patients with MS typically havea higher number of immune cells than a healthy person, which suggeststhat an immune response might play a role. The most common theoriespoint to a virus or genetic defect, or a combination of both. There alsoappears to be a genetic link to the disease. MS is more likely to occurin northern Europe, the northern United States, southern Australia, andNew Zealand than in other areas. Geographic studies indicate there maybe an environmental factor involved. People with a family history of MSand those who live in a geographical area with a higher incidence ratefor MS have a higher risk of the disease.

There is no known cure for multiple sclerosis at this time. However,there are a number of therapies that may slow the disease. The goal oftreatment is to control symptoms and maintain a normal quality of life.

SUMMARY OF THE INVENTION

The present invention provides proteins with glutaminyl cyclaseactivities that constitute novel members of a family of proteins relatedto glutaminyl cyclase, including the full-length proteins, alternativesplice forms, subunits, and mutants, as well as nucleotide sequencesencoding the same. The present invention also provides methods ofscreening for substrates, interacting proteins, agonists, antagonists orinhibitors of the above proteins, and furthermore to pharmaceuticalcompositions comprising the proteins and/or mutants, derivatives and/oranalogues thereof and/or ligands thereto.

These novel proteins having significant sequence similarity toglutaminyl cyclase (nucleic acid sequence of SEQ ID NO 1, proteinsequence of SEQ ID NO 10) are proteins (QPCTLs) from human (furthernamed as human isoQC) (GenBank accession no. NM_(—)017659), mouse(GenBank accession no. NM_(—)027455), Macaca fascicularis (GenBankaccession no. AB168255), Macaca mulatta (GenBank accession no.XM_(—)001110995), cat (GenBank accession no. XM_(—)541552), rat (GenBankaccession no. XM_(—)001066591), cow (GenBank accession no. BT026254) oran analogue thereof having at least 50%/75% sequenceidentity/similarity, preferably 70%/85% sequence identity/similarity,more preferably 90%/95% sequence identity/similarity, most preferably99% sequence identity/similarity.

The protein sequences are given in SEQ. ID NOS: 11 to 18. Furtherdisclosed are nucleic acid sequences coding for these proteins (SEQ. IDNOS: 2 to 9). Table 1 illustrates the similarity between the novelproteins and the known glutaminyl cyclase. Table 2 illustrates theidentity between the novel proteins and the known glutaminyl cyclase.

TABLE 1 Similarity of the protein sequences of the novelglutaminyl-peptide cyclotransferase-like proteins with glutaminylcyclase human isoQC human QC QPCTL source (SEQ ID NO 11) (SEQ ID NO 10)human isoQC — 71.98% (SEQ ID NO 11) M_fascicularis 99.48% 72.24% (SEQ IDNO 13) M_mulatta 99.48% 72.24% (SEQ ID NO 14) C_familiaris 95.82% 72.31%(SEQ ID NO 15) R_norvegicus 95.30% 70.77% (SEQ ID NO 16) M_musculus95.04% 70.77% (SEQ ID NO 17) B_taurus 96.08% 72.31% (SEQ ID NO 18)

TABLE 2 Identity of the protein sequences of the novelglutaminyl-peptide cyclotransferase-like proteins with glutaminylcyclase human isoQC human QC QPCTL source (SEQ ID NO 11) (SEQ ID NO 10)human isoQC — 45.24% (SEQ ID NO 11) M_fascicularis 98.17% 44.99% (SEQ IDNO 13) M_mulatta 98.17% 44.99% (SEQ ID NO 14) C_familiaris 88.51% 45.13%(SEQ ID NO 15) R_norvegicus 84.33% 45.38% (SEQ ID NO 16) M_musculus84.07% 44.62% (SEQ ID NO 17) B_taurus 84.60% 45.64% (SEQ ID NO 18)

There is a high similarity of 95 to 99% and a high identity of 84 to 98%between the QPCTLs from different sources (see FIG. 2). On the basis ofsequence similarity with human and murine glutaminyl cyclase (see FIG.1), one might predict that these QPCTLs would have functions thatinclude, but are not limited to, roles as enzymes. Cloning, expression,biochemical and molecular characterization have confirmed thishypothesis.

The expression pattern of the QPCTLs in brain, prostate and lung tissueis consistent with a role in the diseases described below. The enzymaticactivity as glutaminyl cyclase demonstrates that QPCTLs-activating orinhibiting molecules will have numerous therapeutic applications asdescribed below.

QPCTL activities described herein and their expression patterns arecompatible with their functional roles as physiological regulators ofthe immune and neuroendocrine systems through the enzymatic modificationof biochemical mediators like hormones, peptides and chemokines. Thenumerous functions previously described for QC based upon the use ofinhibitors may be due in part to its action and that of similarproteins, like the QPCTLs. Therefore, the discovery of selective andpotent inhibitors of QC, of the QPCTLs and of other related enzymes isconsidered central to achieving effective and safe pharmaceutical use ofthese and any newly identified glutaminyl-peptide cyclotransferases, aswell as other active compounds that modify the function(s) of suchproteins.

The invention thus provides novel proteins or polypeptides, the nucleicacids coding therefore, cells which have been modified with the nucleicacid so as to express these proteins, antibodies to these proteins, ascreening method for the discovery of new therapeutic agents which areinhibitors of the activity of these proteins (or which are inhibitors ofQC and not of the proteins), and therapeutic agents discovered by suchscreening methods. The novel proteins and the nucleic acids codingtherefore can be used to discover new therapeutic agents for thetreatment of certain diseases, such as for example, neurodegenerative,reproductive, inflammatory and metabolic disorders and also in thepreparation of antibodies with therapeutic or diagnostic value.

In accordance with one aspect of the present invention, there areprovided novel, mature, biologically active proteins, preferably ofhuman origin. Such proteins may be isolated in small quantities fromsuitable animal (including human) tissue or biological fluids bystandard techniques; however, larger quantities are more convenientlyprepared in cultures of cells genetically modified so as to express theprotein.

In accordance with another aspect of the present invention, there areprovided isolated nucleic acid molecules encoding polypeptides of thepresent invention including mRNAs, DNAs, cDNAs, genomic DNAs thereof.

In accordance with a further aspect of the present invention, nucleicacid probes are also provided comprising nucleic acid molecules ofsufficient length to specifically hybridize to a nucleic acid sequenceof the present invention.

In accordance with a still further aspect of the present invention,processes utilizing recombinant techniques are provided for producingsuch polypeptides useful for in vitro scientific research, for example,synthesis of DNA and manufacture of DNA vectors. Processes for producingsuch polypeptides include culturing recombinant prokaryotic and/oreukaryotic host cells that have been transfected with DNA vectorscontaining a nucleic acid sequence encoding such a polypeptide and/orthe mature protein under conditions promoting expression of such proteinand subsequent recovery of such protein or a fragment of the expressedproduct.

In accordance with still another aspect, the invention provides methodsfor using QPCTL polypeptides and polynucleotides for the treatment ofdiseases.

In accordance with yet another aspect of the present invention, there isprovided a process for utilizing such polypeptides, or polynucleotidesencoding such polypeptides, for the discovery of compounds that inhibitthe biological activity of the mature proteins, e.g. the QC activity orthe EC activity, and such inhibitors are thus also provided.

In accordance with a more specific aspect, the invention provides anisolated nucleic acid which encodes (a) a QPCTL polypeptide, selectedfrom SEQ ID NOS: 11 to 18, or (b) having an amino acid sequence that isat least about 75% similar thereto and exhibits the same biologicalfunction, or which is an alternative splice variant of one of SEQ IDNOS: 2 to 9, or which is a probe comprising at least 14 contiguousnucleotides from said nucleic acid encoding (a) or (b), or which iscomplementary to any one of the foregoing.

In accordance with another specific aspect, the invention provides apolypeptide which may be optionally glycosylated, and which (a) has theamino acid sequence of a mature protein set forth in any one of SEQ IDNOS: 10 to 18; preferably of a mature protein set forth in any one ofSEQ ID NOS: 11 to 18 (b) has the amino acid sequence of a mature proteinhaving at least about 75% similarity to one of the mature proteins of(a) and which exhibits the same biological function; (c) has the aminoacid sequence of a mature protein having at least about 50% identitywith a mature protein of any of SEQ ID NOS: 10 to 18; preferably of amature protein set forth in any one of SEQ ID NOS: 11 to 18 or (d) is animmunologically reactive fragment of (a).

In accordance with still another specific aspect, the invention providesa method of screening for a compound capable of inhibiting the enzymaticactivity of at least one mature protein according to the presentinvention, preferably selected from the proteins of SEQ ID NOS: 11 to18, which method comprises incubating said mature protein and a suitablesubstrate for said mature protein in the presence of one or more testcompounds or salts thereof, measuring the enzymatic activity of saidmature protein, comparing said activity with comparable activitydetermined in the absence of a test compound, and selecting the testcompound or compounds that reduce the enzymatic activity.

Further, the present invention pertains to diagnostic kits and methodsbased on the use of a QC-inhibitor, selective QC-inhibitor or selectiveQPCTL-inhibitor.

These and other aspects of the present invention should be apparent tothose skilled in the art from the detailed description, which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the sequence alignment of human QC (hQC), human isoQC(hisoQC), murine QC (mQC) and murine isoQC (misoQC). Multiple sequencealignment was performed using ClustalW at PBIL (Pôe BioinformatiqueLyonnais) (http://npsa-pbil.ibcp.fr) with default settings. Theconservation of the zinc-ion ligating residues is shown for human QC(hQC; GenBank X71125, SEQ ID NO: 10), human isoQC (hisoQC, GenBankNM_(—)017659, SEQ ID NO: 11), murine QC (mQC, GenBank NM_(—)027455, SEQID NO: 79) and murine isoQC (misoQC, GenBank BC058181, SEQ ID NO: 17) inbold and underlined.

FIG. 2 shows the sequence alignment of isoQC from Homo sapiens (hisoQC,GenBank NM_(—)017659, SEQ ID NO: 11), Macaca fascicularis(M_fascicularis, GenBank AB168255, SEQ ID NO: 13), Macaca mulatta(M_mulatta, GenBank XM_(—)001110995, SEQ ID NO: 14), Canis familiaris(C_familiaris, GenBank XM_(—)541552, SEQ ID NO: 15), Rattus norvegicus(R_norvegicus, GenBank XM_(—)001066591, SEQ ID NO: 16), Mus musculus(M_musculus, GenBank BC058181, SEQ ID NO: 17) and Bos taurus (B_taurus,GenBank BT026254, SEQ ID NO: 18). Multiple sequence alignment wasperformed using ClustalW at PBIL (Pôle Bioinformatique Lyonnais)(http://npsa-pbil.ibcp.fr) with default settings. The amino acids of theconserved zinc-ion ligating residues are underlined and typed in bold.

FIG. 3 shows the sequence alignment of human QC (hQC, SEQ ID NO: 10) andhuman isoQC (hisoQC, SEQ ID NO: 12) and other M28 family members of themetallopeptidase Clan MH. Multiple sequence alignment was performedusing ClustalW at ch.EMBnet.org with default settings. The conservationof the amino acid residues ligating the single zinc-ion within the humanQC (hQC; Swiss-Prot Q16769, SEQ ID NO: 10), is shown for the human isoQC(isoQC; Swiss-Prot Q53HE4, SEQ ID NO: 12) (residues 19-382), theZn-dependent aminopeptidase from Streptomyces griseus (SGAP; Swiss-ProtP80561, SEQ ID NO: 80) and the mature Zn-dependent leucyl-aminopeptidasefrom Vibrio proteolyticus (VpAP; Swiss-Prot Q01693, SEQ ID NO: 81). Therespective amino acid residues are underlined and typed in bold.

FIG. 4 shows the sequence alignment of human QC (hQC, SEQ ID NO: 10) andhuman isoQC (hisoQC, SEQ ID NO: 11), showing two putative tranlationalstarts (methionine I—bold, underlined; methionine II—bold). Multiplesequence alignment was performed using ClustalW at PBIL (PôleBioinformatique Lyonnais) http://npsa-pbil.ibcp.fr with defaultsettings. The transmembrane domain, present in human isoQC, is indicatedby the black bar.

FIG. 5 shows the sequence alignment of human QC (hQC, SEQ ID NO: 10) andhuman isoQC (hisoQC, SEQ ID NO: 12), starting with methionine II (bold).Multiple sequence alignment was performed using ClustalW atch.EMBnet.org with default settings. The amino acids involved in metalbinding are underlined and typed in bold. The transmembrane domain,present in human isoQC, is indicated by the black bar.

FIG. 6 shows the analysis of isoQC expression by RT-PCR. Detection inSH-SY5Y, LN405, HaCaT and Hep-G2.

Lanes: bp, DNA standard; 1, amplified PCR product of human isoQC fromSH-SY5Y; 2, amplified PCR product of human isoQC from LN405; 3,amplified PCR product of human isoQC from HaCaT; 4, amplified PCRproduct of human isoQC from Hep-G2.

FIG. 7 shows the analysis of isoQC (Met I, SEQ ID NO: 11) subcellularlocalization by immunhistochemistry. Human isoQC starting at methionineI (see FIG. 5) was expressed as a fusion protein with EGFP (isoQC (MetI)EGFP) in LN 405. Mannosidase II counterstaining was performed usingAB3712 (Chemicon). Merge represents the overlay of isoQC (MetI)-EGFP andMannosidase II staining.

FIG. 8 shows the analysis of isoQC (Met I, SEQ ID NO: 11) subcellularlocalization by immunhistochemistry. Human isoQC starting at methionineI was expressed as a fusion protein with EGFP (isoQC (MetI) EGFP) in LN405. Mitochondrial counterstaining was performed using MAB1273(Chemicon). Merge represents the overlay of isoQC (MetI)-EGFP andmitochondrial staining.

FIG. 9 shows the analysis of isoQC (Met II, SEQ ID NO: 12) subcellularlocalization by immunhistochemistry. Human isoQC starting at methionineII was expressed as a fusion protein with EGFP (isoQC (MetII) EGFP) inLN 405. Mannosidase II counterstaining was performed using AB3712(Chemicon). Merge represents the overlay of isoQC (MetII)-EGFP andMannosidase II staining.

FIG. 10 shows the analysis of isoQC (Met II, SEQ ID NO: 12) subcellularlocalization by immunhistochemistry. Human isoQC starting at methionineII was expressed as a fusion protein with EGFP (isoQC (MetII) EGFP) inLN 405. Mitochondrial counterstaining was performed using MAB1273(Chemicon). Merge represents the overlay of isoQC (MetII)-EGFP andmitochondrial staining.

FIG. 11 shows the analysis of the subcellular localization of isoQC (MetI, SEQ ID NO: 11) by immunhistochemistry. Human isoQC starting atmethionine I was expressed as a fusion protein with EGFP (isoQC (MetI)EGFP) in COS-7. Mannosidase II counterstaining was performed usingAB3712 (Chemicon). Merge represents the overlay of isoQC (MetI)-EGFP andMannosidase II staining.

FIG. 12 shows the analysis of isoQC (Met I, SEQ ID NO: 11) subcellularlocalization by immunhistochemistry. Human isoQC starting at methionineI was expressed as a fusion protein with EGFP (isoQC (MetI) EGFP) inCOS-7. Mitochondrial counterstaining was performed using MAB1273(Chemicon). Merge represents the overlay of isoQC (MetI)-EGFP andmitochondrial staining.

FIG. 13 shows the analysis of isoQC (Met II, SEQ ID NO: 12) subcellularlocalization by immunhistochemistry. Human isoQC starting at methionineII was expressed as a fusion protein with EGFP (isoQC (MetII) EGFP) inCOS-7. Mannosidase II counterstaining was performed using AB3712(Chemicon). Merge represents the overlay of isoQC (MetII)-EGFP andMannosidase II staining.

FIG. 14 shows the analysis of isoQC (Met II, SEQ ID NO: 12) subcellularlocalization by immunhistochemistry. Expression of human isoQC startingat methionine II as a fusion protein with EGFP (isoQC (MetII) EGFP) inCOS-7. Mitochondrial counterstaining was performed using MAB1273(Chemicon). Merge represents the overlay of isoQC (MetII)-EGFP andmitochondrial staining.

FIG. 15 shows the inhibition of human isoQC-catalyzed conversion ofH-Gln-AMC into pGlu-AMC by the inhibitor P150/03. The data wereevaluated according to the Michaelis-Menten kinetic model consideringlinear competitive inhibition. Inhibitor concentrations were as follows:

0 μM

0.3125 μM

0.625 μM

1.25 μM

2.5 μM

5 μM

The determined K_(i)-value was 240±8 nM.

FIG. 16 shows the human isoQC-catalyzed conversion of H-Gln-Ala-OH intopGlu-Ala-OH determined using a spectrophotometric assay. The data wereevaluated according to Michaelis-Menten kinetics. The kinetic parameterswere 324±28 μM and 7.4±0.2 nM/min for the K_(M) and V_(max)-value,respectively.

FIG. 17 provides a schematic representation of the human isoQC proteinconstructs that were expressed hetereologously in the yeast P. pastoris.Two mutations were introduced in some proteins, leading to aglycosylation site at position 55 (155N) and a mutated cystein residueat position 351 (C351A). For expression, the N-terminus including thetransmembrane domain was replaced by a secretion signal of yeast (YSS).The constructs containing the N-terminal secretion signal should beefficiently secreted into the medium.

FIG. 18 shows the QC activity, which was determined in the medium ofexpressing yeast cells. Due to the transmembrane domain, the nativeconstructs were not secreted into the medium (not implemented). Causedby glycosylation (155N), proteins are most efficiently secreted. Themutation C351A resulted also in higher QC activity detected in themedium. The constructs are described in FIG. 17.

FIG. 19 shows the purification of the human isoQC, based on constructYSShisoQCI55NC351A C-His, from the medium of a transgenic P. pastorisstrain. The QC was purified by a combination of IMAC (immobilized metalaffinity chromatography, lane 3), HIC (hydrophobic interactionchromatography, lane 4) and desalting (lane 5). The glycosylation of theenzyme was evidence by enzymatic deglycosalytion, which results in ashift in migration of the protein (lane 6). Lane 1, protein standard:Lane 2, medium prior to purification.

FIG. 20 shows the purification of the human isoQC, based on constructGST-hisoQC C-His, from the cell homogenate of transformed E. coli. TheisoQC was purified by a combination of IMAC (immobilized metal affinitychromatography, lane 3), GST-affinity (lane 4), desalting (lane 5) andion exchange chromatography (lane 6). Lane 1, protein standard: Lane 2,cell homogenate prior to purification. The difference in the molecularmass between the hisoQC which was expressed in yeast and E. coli iscaused by the N-terminal GST-tag fusion. The expressed construct isprovided schematically in the upper part of the figure.

FIG. 21 shows the specificity constants for conversion ofdipeptide-surrogates, dipeptides and oligopeptides by human isoQC(YSShisoQCI55NC351A C-His; compare FIG. 17), GST-hisoQC and human QC.The specificity of GST-hisoQC was the lowest, followed byYSShisoQCI55NC351A C-His. The highest specificity displayed human QC,indicating a higher overall enzymatic activity.

FIG. 22 shows the pH-dependency of catalysis, investigated with humanisoQC (hisoQC), which was expressed in yeast, and human QC (hQC). Bothproteins display a pH-optimum between pH 7 and 8. The fitted curve isbased on three dissociating groups that influence catalysis, one atacidic pH, two at basic pH.

FIG. 23 shows the analysis of conversion of glutamic acid, which ispresent at the N-terminus of the amyloid-β related peptide Aβ(3-11). Theanalysis was performed using Maldi-T of mass spectrometry, the substrateand product differ in their molecular mass/charge ratio of the singlechared molecule by about 18 Da, which is the mass of the released water.In both cases, the same protein concentration was present in thesamples, clearly suggesting that human isoQC also converts N-terminalglutamic acid, but slower than the human QC.

FIG. 24 shows the tissue distribution of murine QC (mQC, SEQ ID NO: 79)and its isoenzyme misoQC (SEQ ID NO: 17), analyzed using real-time PCR.Both the enzymes are expressed in the tested organs. However, theexpression level of mQC was higher in the brain compared with theperipheral organs. In contrast, misoQC was expressed in all testedorgans and tissues at a more similar level, indicating a ubiquitous,“house-keeping” protein.

FIG. 25 shows the time-dependent inhibition of human isoQC (hisoQC) bymetal-chelating compounds 1,10-phenanthroline (circles) and EDTA(squares). Residual hisoQC activity was determined directly afteraddition (closed symbols) or preincubation of hisoQC with respectivereagent for 15 min at 30° C. (open symbols).

FIG. 26 shows the biochemical analysis of the subcellular localizationof QC activity after expression of pcDNA and the native enzymes hisoQC(Met I, SEQ ID NO: 11), hisoQC (Met II, SEQ ID NO: 12) and hQC (SEQ IDNO: 10) in HEK293 cells. (A) specific activity within the cell fractionsin μmole/min/g. (B) absolute activity in nM/min. (C) Expression ofh-isoQC (Met I, SEQ ID NO: 11), h-isoQC (Met II, SEQ ID NO: 12) and hQC(SEQ ID NO: 10) possessing a C-terminal FLAG-tag in HEK293 in comparisonto vector-transfected control (pcDNA), followed by Western Blot analysisapplying specific antibodies detecting either the FLAG-epitope(anti-DYKDDDDK-antibody, Cell Signaling), a 65 kDa protein of humanmitochondria (anti-human mitochondria, Chemicon) or humanSialyltransferase ST1GAL3 (Abnova).

FIG. 27 shows the subcellular localization of human isoQC (hisoQC)signal sequences (A) methionine I—serine 53 and (B) methionine II—serine53, fused to EGFP. Golgi complex was stained using an anti-mannosidaseII antibody and mitochondria were stained using an antibody detecting a65 kDA protein of human mitochondria. Co-localization is shown bysuperimposition of EGFP fluorescence and Red X fluorescence (Merge).

FIG. 28 shows the domain structure of human isoQC (hisoQC) and murineisoQC (misoQC) in comparison to published sequences of humanglycosyltransferases: alpha-N-acetylgalactosaminide alpha-2,6-sialyltransferase 1 (ST6GalNAC1; E.C. 2.4.99.3);beta-1,4-galactosyltransferase 1 (b4Gal-T1, E.C. 2.4.1.-); Galactoside3(4)-L-fucosyltransferase (FucT-III; E. C. 2.4.1.65) andGlycoprotein-fucosylgalactoside alpha-N-acetylgalactosaminyl transferase(NAGAT, E.C.2.4.1.40). The number of amino acids as listed below thecolumns. The cytosolic part is shaded, the transmembrane helix is blackand luminal part is illustrated in white.

FIG. 29 shows the quantification of human isoQC (QPCTL) mRNA indifferent carcinoma cell lines. The QPCTL expression was normalized to50 ng total-RNA. The black bar within the boxes represents therespective median.

FIG. 30 shows the quantification of human isoQC (QPCTL) mRNA expressionin different melanoma cell lines. The QPCTL expression was normalized to50 ng total-RNA.

FIG. 31 shows the quantification of human isoQC (QPCTL) mRNA expressionin samples from soft tissue carcinoma, gastric carcinoma and thyroidcarcinoma from different patients. The QPCTL expression was normalizedto 50 ng total-RNA. The black bar within the boxes represents therespective median.

FIG. 32 shows the human isoQC (QPCTL) mRNA expression in differentgastric carcinomas against their stage of differentiation. QPCTLexpression was normalized to 50 ng total-RNA. The black bar within theboxes represents the respective median.

FIG. 33 shows a comparison of human QC (QPCT) mRNA expression indifferent thyroid carcinomas. QPCT expression was normalized to 50 ngtotal-RNA. The black bar within the boxes represents the respectivemedian. (FTC: folicular thyroid carcinoma; PTC: papillary thyroidcarcinoma; UTC: undifferentiated thyroid carcinoma).

FIG. 34 shows a comparison of human isoQC (QPCTL) mRNA expression indifferent thyroid carcinomas. QPCTL expression was normalized to 50 ngtotal-RNA. The black bar within the boxes represents the respectivemedian. (FTC: folicular thyroid carcinoma; PTC: papillary thyroidcarcinoma; UTC: undifferentiated thyroid carcinoma).

FIG. 35 shows the influence of different stimuli on mRNA expression ofhuman QC (QPCT), human isoQC (QPCTL) and CCL2 in HEK293 cells. Theamount of transcripts is depicted relating to basal expression withoutstimulus. The used concentration of stimulus is stated on the x-axisdrawing.

FIG. 36 shows the influence of different stimuli on mRNA expression ofhuman QC (QPCT), human isoQC (QPCTL) and CCL2 in FTC-133 cells. Theamount of transcripts is depicted relating to basal expression withoutstimulus. The used concentration of stimulus is stated on the x-axisdrawing.

FIG. 37 shows the influence of different stimuli on mRNA expression ofhuman QC (QPCT), human isoQC (QPCTL) and CCL2 in THP-1 cells. The amountof transcripts is depicted relating to basal expression withoutstimulus. The used concentration of stimulus is stated on the x-axisdrawing.

FIG. 38 shows the influence of different stimuli on mRNA expression ofhuman QC (QPCT), CCL2, CCL7, CCL8 and CCL13 in THP-1 cells. The amountof transcripts is depicted relating to basal expression withoutstimulus. The used concentration of stimulus is stated on the x-axisdrawing.

FIG. 39 shows the influence of hypoxia on the mRNA level of human QC(QPCT), human isoQC (QPCTL) and HIF1α in HEK293 (A), FTC-133 (b) andTHP-1 (C).

LIST OF SEQUENCES

SEQ ID NO Description 1 human QC, nucleic acid 2 human isoQC Met I,nucleic acid 3 human isoQC Met II, nucleic acid 4 Macaca fascicularisQPCTL, nucleic acid 5 Macaca mulatta QPCTL, nucleic acid 6 Canisfamiliaris QPCTL, nucleic acid 7 rat QPCTL, nucleic acid 8 mouse QPCTL,nucleic acid 9 bovine QPCTL, nucleic acid 10 human QC, protein 11 humanisoQC Met I, protein 12 human isoQC Met II, protein 13 Macacafascicularis QPCTL, protein 14 Macaca mulatta QPCTL, protein 15 Canisfamiliaris QPCTL, protein 16 rat QPCTL, protein 17 mouse QPCTL, protein18 bovine QPCTL, protein 19 human isoQC splice form 1, nucleic acid 20human isoQC splice form 2, nucleic acid 21 human isoQC splice form 1,protein 22 human isoQC splice form 2, protein 23 Amyloid beta peptide(Abeta) (1-42) 24 Abeta (1-40) 25 Abeta (3-42) 26 Abeta (3-40) 27 Abeta(11-42) 28 Abeta (11-40) 29 pGlu³-Abeta (3-42) 30 pGlu³/-Abeta (3-40) 31pGlu³-Abeta (11-42) 32 pGlu-³-Abeta (11-40) 33 ABri 34 ADan 35 Gastrin17 36 Gastrin 34 37 pGlu-Abri 38 pGlu-ADan 39 pGlu-Gastrin 17 40pGlu-Gastrin 34 41 Neurotensin 42 GnRH 43 CCL16 44 CCL8 45 CCL2 46 CCL1847 Fractalkine 48 CCL7 49 Orexin A 50 Substance P 51 QYNAD 52 pGlu-YNAD53 human isoQC forward primer used for cell line screening 54 humanisoQC reverse primer used for cell line screening 55 forward primer usedfor isolation of human isoQC 56 reverse primer used for isolation ofhuman isoQC 57 forward primer used for cloning of human isoQC (isoformMet I) into vector pEGFP-N3 58 forward primer used for cloning of humanisoQC (isoform Met II) into vector pEGFP-N3 59 reverse primer used forcloning of human isoQC (isoforms Met I and Met II) into vector pEGFP-N360 forward primer used for cloning of human isoQC into vector pET41a 61reverse primer used for cloning of human isoQC into vector pET41a 62forward primer for cloning human isoQC into vector pPICZαA with a C-terminal histidine tag 63 forward primer for cloning human isoQC intovector pPICZaA with a N-terminal histidine tag 64 reverse primer forcloning human isoQC into vector pPICZαA with a N- terminal histidine tag65 forward primer for real-time PCR analysis of isoQC 66 reverse primerfor cloning human isoQC into vector pPICZαA with a C- terminal histidinetag 67 reverse primer for real-time PCR analysis of isoQC 68 Forwardprimer for cloning of murine isoQC cDNA 69 Reverse primer for cloning ofmurine isoQC cDNA 70 Forward primer for cloning of murine isoQC cDNA 71forward primer for real-time PCR analysis of murine QC 72 reverse primerfor real-time PCR analysis of murine QC 73 forward primer for real-timePCR analysis of murine QC 74 reverse primer for real-time PCR analysisof murine QC 75 forward primer for site-directed mutagenesis hisoQC I55N76 reverse primer for site-directed mutagenesis hisoQC I55N 77 forwardprimer for site-directed mutagenesis hisoQC C351A 78 reverse primer forsite-directed mutagenesis hisoQC C351A 79 Mouse glutaminyl cyclaseprotein 80 Streptomyces griseus SGAP 81 Vibrio proteolyticus VpAP 82forward primer for insertion of native hQC into pcDNA 3.1 83 reverseprimer for insertion of native hQC into pcDNA 3.1 84 reverse primer foramplification of hisoQC including the stop codon for insertion intopcDNA 3.1 85 forward primer for amplification EGFP 86 reverse primer foramplification EGFP 87 Reverse primer for amplification of hisoQCN-terminal sequence for fusion with EGFP 88 Reverse primer foramplification hQC C-FLAG for insertion into pcDNA 3.1 89 Reverse primerfor amplification hisoQC C-FLAG for insertion into pcDNA 3.1

DETAILED DESCRIPTION OF THE INVENTION

In accordance with an aspect of the present invention, there areprovided isolated nucleic acid sequences (polynucleotides) of SEQ IDNOS: 2 to 9, 19 and 20, which encode the mature polypeptides having thededuced amino acid sequences of the QPCTLs from different sources (SEQID NOS: 11 to 18, 21 and 22).

Preferred according to the present invention are isolated nucleic acidsequences (polynucleotides) of SEQ ID NOS: 2 and 3, 19 and 20, whichencode the mature polypeptides having the deduced amino acid sequencesof the QPCTLs from human (SEQ ID NOS: 11 and 12, 21 and 22).

More preferred according to the present invention are isolated nucleicacid sequences (polynucleotides) of SEQ ID NOS: 2 and 3, which encodethe mature polypeptides having the deduced amino acid sequences of thehuman QPCTLs of SEQ ID NOS: 11 and 12.

Even preferred according to the present invention are isolated nucleicacid sequences (polynucleotides) of SEQ ID NOS: 19 and 20, which encodethe mature polypeptides having the deduced amino acid sequences ofalternative spliceforms of human QPCTLs of SEQ ID NOS: 21 and 22.

Most preferred according to the present invention is the isolatednucleic acid sequence (polynucleotide) of SEQ ID NO: 2, which encodesthe mature polypeptide having the deduced amino acid sequence of thehuman QPCTL of SEQ ID NOS: 11.

Even most preferred according to the present invention is the isolatednucleic acid sequence (polynucleotide) of SEQ ID NO: 3, which encodesthe mature polypeptide having the deduced amino acid sequence of thehuman QPCTL of SEQ ID NOS: 12.

The aforementioned embodiments and preferences apply to the QPCTLnucleic acids as well as QPCTL proteins and any desired method of use,diagnosing, treatment, screening, effectors, inhibitors and other usesand methods according to the present invention.

The polynucleotides of this invention were discovered by similaritysearch using Nucleotide BLAST at NCBI(http://www.ncbi.nlm.nih.gov/BLAST/) applying human QC as template. Thesearch resulted in discovery of a putative QPCTL on chromosome 19, whichis encoded in region 19q13.32. On basis of the search, primers for acell line screening of human isoQC were designed (Table 4). The isolatedcDNA for human QPCTL contains an open reading frame encoding a proteinof 382 amino acids in length, which is related to human QC displaying45.24% sequence identity, and 71.98% similarity. Applying differentbioinformatic algorithms (www.expasy.ch) for prediction of thesubcellular localization did not result in a reliable result. Theprognosis, depending on the prediction program, was transfer togolgi-apparatus or mitochondria.

Amino acid sequence alignments of human QPCTL with other members of theM28 family members of the metallopeptidase Clan MH shows that humanQPCTL protein has overall sequence and structural homology to human andmurine QC (FIG. 1) and bacterial aminopeptidases (FIG. 3). A databasesearch for additional human QPCTL-related genes revealed the presence ofrodent, simian, cattle and dog QPCTLs. Alignment of these sequences withthe novel human QPCTL shows that they display considerable homology withits human counterpart. The zinc-complexing residues of human QC(Asp-Glu-His) are conserved within QPCTLs from the different origins(FIG. 2).

The human isoQC gene contains at least 8 exons. The sequence coding forthe human isoQC protein is located on exons 1 to 7. Human isoQC maps tochromosome 19 at position 19q13.32. A cell line screening for humanisoQC revealed transcripts in cells origin from liver (Hep-G2,hepatocellular carcinoma), skin (HaCaT, keratinocyte) and neuronaltissues (LN405, astrocytoma; SH-SY5Y, neuroblastoma) (FIG. 6).

The isolated QPCTL-cDNA was tested on functional expression in severalexpression hosts. Expression in P. pastoris, which was successfullyapplied for human QC, did not result in an enzymatically active protein.Expression in mammalian cells resulted in detection of activity,however, expression levels were very low. Thus, the isolation of anenzymatically active protein was not possible with the knowledge of theskilled artisan. Enzymatically active protein was isolated onlyfollowing expression of a GST-QPCTL fusion protein in E. coli, applyingvery unusual expression conditions: Expression for 4 h at 37° C. inpresence of 1% Glucose, induction of expression using 20 μM IPTG.

The expression conditions result in a low-level expression in E. coli,which is necessary for functional folding of the peptide chain.

In another embodiment, the present invention relates to QPCTL knockoutanimals, preferably rats or mice. The use of knockout mice in furtheranalysis of the function of QPCTL genes is a valuable tool.

The polynucleotides of the present invention may be in the form of RNAor in the form of DNA; DNA should be understood to include cDNA, genomicDNA, and synthetic DNA. The DNA may be double-stranded orsingle-stranded and, if single stranded, may be the coding strand ornon-coding (antisense) strand. The coding sequence, which encodes themature polypeptide may be identical to the coding sequence shown in SEQID NOS 2 to 9, or it may be a different coding sequence encoding thesame mature polypeptide, as a result of the redundancy or degeneracy ofthe genetic code or a single nucleotide polymorphism. For example, itmay also be an RNA transcript which includes the entire length of anyone of SEQ ID NOS 11 to 18.

The polynucleotides which encode the mature proteins of SEQ ID NOS 2 to9 may include but are not limited to the coding sequence for the matureprotein alone; the coding sequence for the mature polypeptide plusadditional coding sequence, such as a leader or secretory sequence or aproprotein sequence; and the coding sequence for the mature protein (andoptionally additional coding sequence) plus non-coding sequence, such asintrons or a non-coding sequence 5′ and/or 3′ of the coding sequence forthe mature protein.

Thus, the term “polynucleotide encoding a polypeptide” or the term“nucleic acid encoding a polypeptide” should be understood to encompassa polynucleotide or nucleic acid which includes only coding sequence forthe mature protein as well as one which includes additional codingand/or non-coding sequence. The terms polynucleotides and nucleic acidare used interchangeably.

The present invention also includes polynucleotides where the codingsequence for the mature protein may be fused in the same reading frameto a polynucleotide sequence which aids in expression and secretion of apolypeptide from a host cell; for example, a leader sequence whichfunctions as a secretory sequence for controlling transport of apolypeptide from the cell may be so fused. The polypeptide having such aleader sequence is termed a preprotein or a preproprotein and may havethe leader sequence cleaved, by the host cell to form the mature form ofthe protein. These polynucleotides may have a 5′ extended region so thatit encodes a proprotein, which is the mature protein plus additionalamino acid residues at the N-terminus. The expression product havingsuch a prosequence is termed a proprotein, which is an inactive form ofthe mature protein; however, once the prosequence is cleaved an activemature protein remains. Thus, for example, the polynucleotides of thepresent invention may encode mature proteins, or proteins having aprosequence, or proteins having both a prosequence and a presequence(leader sequence).

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptides of the present invention. The markersequence may be a polyhistidine tag, a hemagglutinin (HA) tag, a c-myctag or a V5 tag when a mammalian host, e.g. COS-1 cells, is used.

The HA tag would correspond to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37: 767 (1984)), andthe c-myc tag may be an epitope from human Myc protein (Evans, G. I. etal., Mol. Cell. Biol. 5: 3610-3616 (1985)).

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

The term “significant sequence homology” is intended to denote that atleast 25%, preferably at least 40%, of the amino acid residues areconserved, and that, of the nonconserved residues, at least 40% areconservative substitutions.

Fragments of the full-length genes of the present invention may be usedas a hybridization probe for a cDNA library to isolate full-length cDNAas well as to isolate other cDNAs, which have significant sequencehomology to the gene and will encode proteins or polypeptides havingsimilar biological activity or function. By similar biological activityor function, for purposes of this application, is meant the ability toform pyroglutamate from a N-terminal glutamine or glutamic acid ofpeptides, proteins, hormones or other substrates, defined as QC- andEC-activity, respectively. Such a probe of this type has at least 14bases (at least 14 contiguous nucleotides from one of SEQ ID NOS: 2 to9), preferably at least 30 bases, and such may contain, for example, 50or more bases. Preferred are the probes of SEQ ID NOS 53 to 61. Suchprobe may also be used to identify a cDNA clone corresponding to afull-length transcript and/or a genomic clone or clones that containsthe complete gene, including regulatory and promoter regions, exons, andintrons. Labelled oligonucleotides having a sequence complementary tothat of the gene of the present invention are useful to screen a libraryof human cDNA, genomic DNA or mRNA or similar libraries from othersources or animals to locate members of the library to which the probehybridizes. As an example, a known DNA sequence may be used tosynthesize an oligonucleotide probe, which is then used in screening alibrary to isolate the coding region of a gene of interest.

The present invention is considered to further provide polynucleotideswhich hybridize to the hereinabove-described sequences wherein there isat least about 70%, preferably at least about 90%, more preferably atleast about 95%, and most preferably at least about 99% identity orsimilarity between the sequences, and thus encode proteins havingsimilar biological activity. Moreover, as known in the art, there is“similarity” between two polypeptides when the amino acid sequencescontain the same or conserved amino acid substitutes for each individualresidue in the sequence. Identity and similarity may be measured usingsequence analysis software (e.g., ClustalW at PBIL (Pôle BioinformatiqueLyonnais) http://npsa-pbil.ibcp.fr). The present invention particularlyprovides such polynucleotides, which hybridize under stringentconditions to the hereinabove-described polynucleotides. As herein used,the term “stringent conditions” means conditions which permithybridization between polynucleotides sequences and the polynucleotidesequences of SEQ ID NOS: 2 to 9 where there is at least about 70%identity.

Suitably stringent conditions can be defined by, e.g., theconcentrations of salt or formamide in the prehybridization andhybridization solutions, or by the hybridization temperature, and arewell known in the art. In particular, stringency can be increased byreducing the concentration of salt, by increasing the concentration offormamide, and/or by raising the hybridization temperature.

For example, hybridization under high stringency conditions may employabout 50% formamide at about 37° C. to 42° C., whereas hybridizationunder reduced stringency conditions might employ about 35% to 25%formamide at about 30° C. to 35° C. One particular set of conditions forhybridization under high stringency conditions employs 42° C., 50%formamide, 5×. SSPE, 0.3% SDS, and 200 μg/ml sheared and denaturedsalmon sperm DNA. For hybridization under reduced stringency, similarconditions as described above may be used in 35% formamide at a reducedtemperature of 35° C. The temperature range corresponding to aparticular level of stringency can be further narrowed by calculatingthe purine to pyrimidine ratio of the nucleic acid of interest andadjusting the temperature accordingly. Variations on the above rangesand conditions are well known in the art. Preferably, hybridizationshould occur only if there is at least 95%, and more preferably at least97%, identity between the sequences. The polynucleotides which hybridizeto the hereinabove described polynucleotides in a preferred embodimentencode polypeptides which exhibit substantially the same biologicalfunction or activity as the mature protein encoded by one of the cDNAsof SEQ ID NOS: 2 to 9.

As mentioned, a suitable polynucleotide probe may have at least 14bases, preferably 30 bases, and more preferably at least 50 bases, andwill hybridize to a polynucleotide of the present invention, which hasan identity thereto, as hereinabove described, and which may or may notretain activity. For example, such polynucleotides may be employed as aprobe for hybridizing to the polynucleotides of SEQ ID NOS: 2 to 9respectively, for example, for recovery of such a polynucleotide, or asa diagnostic probe, or as a PCR primer. Thus, the present inventionincludes polynucleotides having at least about a 70% identity,preferably at least about a 90% identity, and more preferably at leastabout a 95% identity, and most preferably at least about a 99% identityto a polynucleotide which encodes the polypeptides of SEQ ID NOS: 11 to18 respectively, as well as fragments thereof, which fragmentspreferably have at least 30 bases and more preferably at least 50 bases,and to polypeptides encoded by such polynucleotides.

As is well known in the art, the genetic code is redundant in thatcertain amino acids are coded for by more than one nucleotide triplet(codon), and the invention includes those polynucleotide sequences whichencode the same amino acids using a different codon from thatspecifically exemplified in the sequences herein. Such a polynucleotidesequence is referred to herein as an “equivalent” polynucleotidesequence. The present invention further includes variants of thehereinabove described polynucleotides which encode for fragments, suchas part or all of the mature protein, analogs and derivatives of one ofthe polypeptides having the deduced amino acid sequence of any one ofSEQ ID NOS: 11 to 18. The variant forms of the polynucleotides may be anaturally occurring allelic variant of the polynucleotides or anon-naturally occurring variant of the polynucleotides. For example, thevariant in the nucleic acid may simply be a difference in codon sequencefor the amino acid resulting from the degeneracy of the genetic code, orthere may be deletion variants, substitution variants and addition orinsertion variants. As known in the art, an allelic variant is analternative form of a polynucleotide sequence, which may have asubstitution, deletion or addition of one or more nucleotides that doesnot substantially alter the biological function of the encodedpolypeptide.

The present invention further includes polypeptides, which have thededuced amino acid sequence of SEQ ID NOS: 11 to 18, as well asfragments, analogs and derivatives of such polypeptides. The terms“fragment”, “derivative” and “analog”, when referring to thepolypeptides of SEQ ID NOS: 11 to 18, means polypeptides that retainessentially the same biological function or activity as suchpolypeptides. An analog might, for example, include a proprotein, whichcan be activated by cleavage of the proprotein portion to produce anactive mature protein. The polypeptides of the present invention may berecombinant polypeptides, natural polypeptides or synthetic polypeptide;however, they are preferably recombinant polypeptides, glycosylated orunglycosylated.

The fragment, derivative or analog of a polypeptide of any one of SEQ IDNOS 11 to 18, may be (i) one in which one or more of the amino acidresidues is substituted with a conserved or non-conserved amino acidresidue (preferably a conserved amino acid residue) and such substitutedamino acid residue may or may not be one encoded by the genetic code, or(ii) one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which additional amino acids arefused to the mature protein, such as a leader or secretory sequence or asequence which is employed for purification of the mature polypeptide ora proprotein sequence. Such fragments, derivatives and analogs aredeemed to be within the scope of those skilled in the art to provideupon the basis of the teachings herein.

The polypeptides and polynucleotides of the present invention should bein an isolated form, and preferably they are purified to substantialhomogeneity or purity. By substantial homogeneity is meant a purity ofat least about 85%.

The term “isolated” is used to mean that the material has been removedfrom its original environment (e.g., the natural environment if it isnaturally occurring). For example, a naturally occurring polynucleotideor polypeptide present in a living animal is not considered to beisolated, but the same polynucleotide or polypeptide, when separatedfrom substantially all of the coexisting materials in the naturalsystem, is considered isolated. For DNA, the term includes, for example,a recombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote; or which exists as a separate molecule (e.g., acDNA or a genomic or cDNA fragment produced by polymerase chain reaction(PCR) or restriction endonuclease digestion) independent of othersequences. It also includes a recombinant DNA, which is part of a hybridgene encoding additional polypeptide sequence, e.g., a fusion protein.Further included is recombinant DNA which includes a portion of thenucleotides shown in one of SEQ ID NOS 2 to 9 which encodes analternative splice variant of the QPCTLs. Various alternative splicevariants are exemplified in SEQ ID NOS: 19-22.

The polypeptides of the present invention include any one of thepolypeptides of SEQ ID NOS 11 to 18 (in particular the mature proteins),as well as polypeptides which have at least 75% similarity (e.g.preferably at least 50% and more preferably at least 70% identity) toone of the polypeptides of SEQ ID NOS 11 to 18, more preferably at least85% similarity (e.g. preferably at least 70% identity) to one of thepolypeptides of SEQ ID NOS 11 to 18, and most preferably at least 95%similarity (e.g. preferably at least 90% identity) to any one of thepolypeptides of SEQ ID NOS 11 to 18. Certain preferred embodiments canhave at least about 95% sequence identity or more, including, forexample, at least about 96% sequence identity, at least about 97%sequence identity, at least about 98% sequence identity, or at leastabout 99% sequence identity. Moreover, they should preferably includeexact portions of such polypeptides containing a sequence of at least 30amino acids, and more preferably at least 50 amino acids.

Fragments or portions of the polypeptides of the present invention maybe employed as intermediates for producing the corresponding full-lengthpolypeptides by peptide synthesis. Fragments or portions of thepolynucleotides of the present invention may also be used to synthesizefull-length polynucleotides of the present invention.

The present invention also includes vectors, which include suchpolynucleotides, host cells which are genetically engineered with suchvectors and the production of polypeptides by recombinant techniquesusing the foregoing. Host cells are genetically engineered (transducedor transformed or transfected) with such vectors, which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the genes of the present invention. Theculture conditions, such as temperature, pH and the like, are thosecommonly used with the host cell selected for expression, as well knownto the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotides may be included in any one of a variety of expressionvectors for expressing polypeptides. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by any of avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site (s) by procedures well knownin the art, which procedures are deemed to be within the scope of thoseskilled in this art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence (s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli lac or trp, the phagelambda P.sub.L promoter and other promoters known to control expressionof genes in prokaryotic or eukaryotic cells or their viruses.

The expression vector should also contain a ribosome binding site fortranslation initiation and a transcription terminator. The vector mayalso include appropriate sequences for amplifying expression. Inaddition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells, such as dihydrofolate reductase orneomycin-resistance for eukaryotic cell culture, or such astetracycline-or ampicillin-resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein. As representative examples of appropriate hosts,there may be mentioned: bacterial cells, such as E. coli, Streptomyces,Salmonella typhimurium; fungal cells, such as yeast; insect cells, suchas Drosophila S2 and Spodoptera Sf9; animal cells, such as CHO, COS orBowes melanoma; adenoviruses; plant cells, etc. The selection of anappropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

Synthetic production of nucleic acid sequences is well known in the artas is apparent from CLONTECH 95/96 Catalogue, pages 215-216, CLONTECH,1020 East Meadow Circle, Palo Alto, Calif. 94303. Thus, the presentinvention also includes expression vectors useful for the production ofthe proteins of the present invention. The present invention furtherincludes recombinant constructs comprising one or more of the sequencesas broadly described above. The constructs may comprise a vector, suchas a plasmid or viral vector, into which a sequence of the invention hasbeen inserted, in a forward or reverse orientation. In a preferredaspect of this embodiment, the construct further comprises regulatorysequences, including, for example, a promoter, operably linked to thesequence. Large numbers of suitable vectors and promoters are known tothose of skill in the art, and are commercially available. The followingvectors are provided by way of example: Bacterial: pQE70, pQE60, pQE-9(Qiagen), pBS, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A,pNH16a, pNHI8A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540and pRIT5 (Pharmacia); and Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG(Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia). However, anyother suitable plasmid or vector may be used as long as it is replicableand viable in the host.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol acetyl transferase) vectors or other vectors withselectable markers.

Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P.sub.R,P.sub.L and trp. Eukaryotic promoters include CMV immediate early, HSVthymidine kinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

Components of the expression vector may generally include: 1) a neomycinphosphotransferase (G418), or hygromycin B phosphotransferase (hyg) geneas a selection marker, 2) an E. coli origin of replication, 3) a T7 andSP6 phage promoter sequence, 4) lac operator sequences, 5) the lactoseoperon repressor gene (lacIq) and 6) a multiple cloning site linkerregion. Such an origin of replication (oriC) may be derived from pUC19(LTI, Gaithersburg, Md.).

A nucleotide sequence encoding one of the polypeptides of SEQ ID NOS: 2to 9 having the appropriate restriction sites is generated, for example,according to the PCR protocol described in Examples 1 and 2 hereinafter,using PCR primers having restriction sites for EcoR I (as the 5′ primer)and Sal I (as the 3′ primer) for cloning of isoQC Met I and Met II intovector EGFP-N3, or sites for Spe I (as the 5′ primer) and EcoR I (as the3′ primer) for cloning of isoQC into vector pET41a. The PCR inserts aregel-purified and digested with compatible restriction enzymes. Theinsert and vector are ligated according to standard protocols.

In a further embodiment, the present invention provides host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, lipofection or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

Such constructs in host cells are preferably used in a conventionalmanner to produce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers or by chemical ligation ofsuitable fragments thus prepared.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989).

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector.

Enhancers include cis-acting elements of DNA, usually about from 10 to300 bp, that act on a promoter to increase its transcription. Examplesinclude the SV40 enhancer on the late side of the replication origin bp100 to 270, acytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes, such as3-phosphoglycerate kinase (PGK), alpha-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter.

The vector will comprise one or more phenotypic selectable markers andan origin of replication to ensure maintenance of the vector and to, ifdesired, provide amplification within the host. Suitable prokaryotichosts for transformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces and Staphylococcus, although others may also be employed asa matter of choice.

As a representative but non-limiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,U.S.A.). These pBR322 “backbone” sections are combined with anappropriate promoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction), and cells are cultured for an additional period.

Cells are typically harvested by centrifugation and then disrupted byphysical or chemical means, with the resulting crude extract beingretained for further purification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption and use of cell-lysing agents; such methods arewell known to those skilled in the art.

Various mammalian cell culture systems can also be employed to express arecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23: 175 (1981). Other cell lines capable of expressing acompatible vector include, for example, the C127, 3T3, CHO, HeLa and BHKcell lines. Mammalian expression vectors will generally comprise anorigin of replication, a suitable promoter and enhancer, and also anynecessary ribosome binding sites, polyadenylation site, splice donor andacceptor sites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide required nontranscribedgenetic elements.

The polypeptides can be recovered and purified from recombinant cellcultures by methods including ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Recovery can be facilitated if the polypeptide isexpressed at the surface of the cells, but such is not a prerequisite.Recovery may also be desirable of cleavage products that are cleavedfollowing expression of a longer form of the polypeptide. Proteinrefolding steps as known in this art can be used, as necessary, tocomplete configuration of the mature protein. High performance liquidchromatography (HPLC) can be employed for final purification steps.

The polypeptides of the present invention may be purified naturalproducts, or produced by recombinant techniques from a prokaryotic oreukaryotic host (for example, by bacterial, yeast, higher plant, insector mammalian cells in culture). Depending upon the host employed in arecombinant production procedure, the polypeptides of the presentinvention may be glycosylated or may be non-glycosylated. Polypeptidesof the invention may also include an initial methionine amino acidresidue.

In a preferred embodiment, the proteins of the invention are isolatedand purified so as to be substantially free of contamination from otherproteins. For example, the proteins of the invention should constituteat least 80% by weight of the total protein present in a sample, morepreferably at least 90%, even more preferably at least 95%, and mostpreferably at least 98% by weight of the total protein.

These proteins may be in the form of a solution in water, anothersuitable solvent, such as dimethyl sulphoxide (DMSO) or ethanol, or amixture of suitable solvents.

Examples of mixtures of solvents include 10% (by weight) ethanol inwater and 2% (by weight) DMSO in water. A solution may further comprisesalts, buffering agents, chaotropic agents, detergents, preservativesand the like. Alternatively, the proteins may be in the form of a solid,such as a lyophilised powder or a crystalline solid, which may alsocomprise a residual solvent, a salt or the like.

As used herein, the term “antibodies” includes polyclonal antibodies,affinity-purified polyclonal antibodies, monoclonal antibodies, andantigen-binding fragments, such as F (ab′)2 and Fab′proteolyticfragments. Genetically engineered intact antibodies or fragments, suchas chimeric antibodies, Fv fragments, single chain antibodies and thelike, as well as synthetic antigen-binding peptides and polypeptides,are also included. Non-human antibodies may be humanized by graftingnon-human CDRs onto human framework and constant regions, or byincorporating the entire non-human variable domains (optionally“cloaking” them with a human-like surface by replacement of exposedresidues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans should be reduced.

Alternative techniques for generating or selecting antibodies usefulherein include in vitro exposure of lymphocytes to human isoQC proteinor a peptide therefrom, and selection of antibody display libraries inphage or similar vectors (for instance, through use of immobilized orlabeled human isoQC protein or peptide).

Genes encoding polypeptides having potential human isoQC polypeptidebinding domains can be obtained by screening random peptide librariesdisplayed on phage (phage display) or on bacteria, such as E. coli.Nucleotide sequences encoding such polypeptides can be obtained in anumber of ways well known in the art.

As would be evident to one of ordinary skill in the art, polyclonalantibodies can be generated from inoculating a variety of warm-bloodedanimals, such as horses, cows, goats, sheep, dogs, chickens, rabbits,mice and rats, with a human isoQC polypeptide or a fragment thereof. Theimmunogenicity of a human isoQC polypeptide may be increased through theuse of an adjuvant, such as alum (aluminum hydroxide) or Freund'scomplete or incomplete adjuvant, or surface active substances, such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLHor dinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.Polypeptides useful for immunization also include fusion polypeptides,such as fusions of isoQC or a portion thereof with an immunoglobulinpolypeptide or with maltose binding protein. The polypeptide immunogenmay be a full-length molecule or a portion thereof. If the polypeptideportion is “hapten-like”, such portion may be advantageously joined orlinked to a macromolecular carrier, such as keyhole limpet hemocyanin(KLH), bovine serum albumin (BSA) or tetanus toxoid, for immunization.Antibodies to isoQC may also be generated using methods that are wellknown in the art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, and single chain antibodies, Fabfragments, and fragments produced by a Fab expression library.

Neutralizing antibodies (i.e., those which block or modify interactionsat the active sites) are especially preferred for therapeutic use.

For the production of antibodies, binding proteins, or peptides whichbind specifically to QPCTL, libraries of single chain antibodies, Fabfragments, other antibody fragments, non-antibody protein domains, orpeptides may be screened. The libraries could be generated using phagedisplay, other recombinant DNA methods, or peptide synthesis (Vaughan,T. J. et al. Nature Biotechnology 14: 309-314 (1966)). Such librarieswould commonly be screened using methods, which are well known in theart to identify sequences which demonstrate specific binding to QPCTL.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to QPCTL have an amino acid sequence consisting of atleast about 5 amino acids and, more preferably, of at least about 10amino acids. It is also preferable that these oligopeptides, peptides,or fragments are identical to a portion of the amino acid sequence ofthe natural protein. Short stretches of QPCTL amino acids may also befused with those of another protein, such as KLH, and antibodies to thechimeric molecule may be produced.

Monoclonal antibodies to QPCTL may be prepared using any well knowntechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique, although monoclonal antibodies produced byhybridoma cells may be preferred.

In addition, techniques developed for the production of “chimericantibodies”, such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used, see Neuberger, M. S. et al. Nature312: 604-608 (1984). Alternatively, techniques described for theproduction of single chain antibodies may be adapted, using methodsknown in the art, to produce QPCTL-specific single chain antibodies.Antibodies with related specificity, but of distinct idiotypiccomposition, may be generated by chain shuffling from randomcombinatorial immunoglobulin libraries. (Burton D. R. Proc. Natl. Acad.Sci. 88: 11120-11123 (1991)).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature.(Orlandi, R. et al. Proc. Natl. Acad. Sci. 86: 3833-3837 (1989)).

Antibody fragments, which contain specific binding sites for QPCTL mayalso be generated. For example, such fragments include, but are notlimited to, F(ab′)₂ fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab′)₂ fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. (Huse, W. D. etal. Science 254: 1275-1281 (1989)).

Various immunoassays may be used to identify antibodies having thedesired specificity. Numerous protocols for competitive binding orimmunoradiometric assays using either polyclonal or monoclonalantibodies with established specificities are well known in the art.Such immunoassays typically involve the measurement of complex formationbetween QPCTL and its specific antibody. A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering QPCTL epitopes is preferred, but a competitive bindingassay may also be employed.

As earlier mentioned, the QPCTLs can be used in treatment of theDiseases.

Pharmaceutical compositions suitable for use in this aspect of theinvention include compositions wherein the active ingredients arecontained in an effective amount to achieve the intended purposerelating to one of the Diseases. The determination of a therapeuticallyeffective dose is well within the capability of those skilled in the artand can be estimated initially either in cell culture assays, e.g. ofneoplastic cells, or in animal models, usually mice, rats, rabbits,dogs, or pigs. An animal model may also be used to determine theappropriate concentration range and route of administration, whichinformation is then commonly used to determine useful doses and routesfor administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, e.g. a QPCTL or fragment thereof, antibodies of DPRP, or anagonist, antagonist or inhibitor of QPCTL, which ameliorates particularsymptoms or conditions of the disease. For example, the amount to beadministered may be effective to cyclise N-terminal Glu or Gln of adesired target substrate upon contact therewith. Therapeutic efficacyand toxicity may likewise be determined by standard pharmaceuticalprocedures in cell cultures or with experimental animals, such as bycalculating the ED50 (the dose therapeutically effective in 50% of thepopulation) or LD50 (the dose lethal to 50% of the population)statistics. The dose ratio of toxic to therapeutic effects is thetherapeutic index, and it can be expressed as the LD50/ED50 ratio.Pharmaceutical compositions, which exhibit large therapeutic indices,are preferred. The data obtained from cell culture assays and animalstudies is used in formulating a range of dosage for human use. Thedosage contained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, the sensitivity of the patient, and the route ofadministration.

An exact dosage will normally be determined by the medical practitionerin light of factors related to the subject requiring treatment, withdosage and administration being adjusted to provide a sufficient levelof the active moiety or to maintain a desired effect.

Factors to be taken into account include the severity of the diseasestate, the general health of the subject, the age, weight, and gender ofthe subject, diet, time and frequency of administration, drugcombination (s), reaction sensitivities, and tolerance/response totherapy. Long-acting pharmaceutical compositions may be administeredevery 3 to 4 days, every week, or even once every two weeks, dependingon the half-life and clearance rate of the particular formulation.

Yet another aspect of the invention provides polynucleotide moleculeshaving sequences that are antisense to mRNA transcripts of apolynucleotide of SEQ ID NOS 2 to 9. Administration of an antisensepolynucleotide molecule can block the production of the protein encodedby the QPCTL genes of SEQ ID NOS 2 to 9. The techniques for preparingantisense polynucleotide molecules and administering such molecules areknown in the art. For example, antisense polynucleotide molecules can beencapsulated into liposomes for fusion with cells.

In particular, the expression of the QPCTL genes of SEQ ID NOS 2 to 9 inbrain, prostate, lung, heart, liver, spleen and kidney tissue providesevidence for a potential role in the pathophysiology of the diseasesdescribed below. Therefore in a further aspect, the invention relates todiagnostic assays for detecting diseases associated with inappropriateQPCTL activity or expression levels. Antibodies that specifically bindQPCTL may be used for the diagnosis of disorders characterized byexpression of QPCTL, or in assays to monitor patients being treated withQPCTL or with agonists or antagonists (inhibitors) of QPCTL. Antibodiesuseful for diagnostic purposes may be prepared in the same manner asthose described above for therapeutics. Diagnostic assays for QPCTLinclude methods that utilize the antibody and a label to detect QPCTL inhuman body fluids or in extracts of cells or tissues. The antibodies maybe used with or without modification, and they may be labeled bycovalent or non-covalent joining with a reporter molecule. A widevariety of reporter molecules are known in the art. Recombinant QPCTLproteins that have been modified so as to be catalytically inactive canalso be used as dominant negative inhibitors. Such modificationsinclude, for example, mutation of the active site.

A variety of protocols for measuring QPCTL, including ELISAs, RIAs andFACS, are known in the art and provide a basis for diagnosing altered orabnormal levels of QPCTL expression. Normal or standard values for QPCTLexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toQPCTL under conditions suitable for complex formation. The method fordetecting QPCTL in a biological sample would comprise the steps of a)providing a biological sample; b) combining the biological sample and ananti-QPCTL antibody under conditions which are suitable for complexformation to occur between QPCTL and the antibody; and c) detectingcomplex formation between QPCTL and the antibody, thereby establishingthe presence of QPCTL in the biological sample.

The amount of complex formation then may be quantified by variousmethods, preferably by photometric means. Quantities of QPCTL expressedin a subject, control, and disease samples from biopsied tissues arecompared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingQPCTL are used for diagnostic purposes, which polynucleotides mayinclude oligonucleotide sequences, complementary RNA and DNA molecules,and PNAs. These polynucleotides may be used to detect and quantitategene expression in biopsied tissues in which expression of QPCTL may becorrelated with one of the diseases. The diagnostic assay may be used todistinguish between absence, presence, and excess expression of QPCTLand to monitor regulation of QPCTL levels during therapeuticintervention. Moreover, pharmacogenomic, single nucleotide polymorphisms(SNP) analysis of the QPCTL genes can be used as a method to screen formutations that indicate predisposition to disease or modified responseto drugs.

QPCTL polynucleotide and polypeptide sequences, fragments thereof,antibodies of QPCTLs, and agonists, antagonists or inhibitors of QPCTLscan be used as discovery tools to identify molecular recognition eventsand therefore proteins, polypeptides and peptides that interact withQPCTL proteins. A specific example is phage display peptide librarieswhere greater than 108 peptide sequences can be screened in a singleround of panning. Such methods as well as others are known within theart and can be utilized to identify compounds that inhibit or enhancethe activity of any one of the QPCTLs of SEQ ID NOS 11-18.

Coupled links represent functional interactions such as complexes orpathways, and proteins that interact with QPCTLs can be identified by ayeast two-hybrid system, proteomics (differential 2D gel analysis andmass spectrometry) and genomics (differential gene expression bymicroarray or serial analysis of gene expression SAGE).

Proteins identified as functionally linked to QPCTLs and the process ofinteraction form the basis of methods of screening for inhibitors,agonists and antagonists and modulators of these QPCTL-proteininteractions.

The term “antagonist”, as it is used herein, refers to an inhibitormolecule which, when bound to QPCTL, decreases the amount or theduration of the effect of the biological or immunological activity ofQPCTL, e.g. decreasing the enzymatic activity of the peptidase tocyclise Glu- or Gln-residues at the N-termini of the QPCTL substrates.Antagonists may include proteins, nucleic acids, carbohydrates,antibodies, or any other molecules which decrease the effect of QPCTL;for example, they may include small molecules and organic compounds thatbind to and inactivate QPCTLs by a competitive or non-competitive typemechanism. Preferred are small molecule inhibitors of QPCTL. Mostpreferred are competitive small molecule inhibitors of QPCTL.

Specific examples of QPCTL enzyme activity inhibitors are described inExample 4. Inhibitors can be, for example, inhibitors of the QPCTLcyclase activity, or alternatively inhibitors of the binding activity ofthe QPCTL to proteins with which they interact. Specific examples ofsuch inhibitors can include, for example, anti-QPCTL antibodies,peptides, protein fragments, or small peptidyl protease inhibitors, orsmall non-peptide, organic molecule inhibitors which are formulated in amedium that allows introduction into the desired cell type.Alternatively, such inhibitors can be attached to targeting ligands forintroduction by cell-mediated endocytosis and other receptor mediatedevents. Such methods are described further below and can be practiced bythose skilled in the art given the QPCTL nucleotide and amino acidsequences described herein.

A further use of QPCTLs is for the screening of potential antagonistsfor use as therapeutic agents, for example, for inhibiting binding toQPCTL, as well as for screening for agonists. QPCTL, its immunogenicfragments, or oligopeptides thereof can be used for screening librariesof compounds which are prospective agonists or antagonists in any of avariety of drug screening techniques. The fragment employed in suchscreening may be free in solution, affixed to a solid support, borne ona cell surface, or located intracellularly. The formation of bindingcomplexes between QPCTL and the agent being tested is then measured.Other assays to discover antagonists that will inhibit QPCTL areapparent from the disclosures of Patents Nos. WO 2004/098625, WO2004/098591 and WO 2005/075436, which describe inhibitors of QC andwhich are incorporated herein in their entirety. Another worthwhile useof these QPCTLs is the screening of inhibitors of QC to show that theywill not have undesired side effects by also inhibiting one or more ofthe QPCTLs.

A method provided for screening a library of small molecules to identifya molecule which binds QPCTL generally comprises: a) providing a libraryof small molecules; b) combining the library of small molecules with thepolypeptide of either SEQ ID NOS 11 to 18, or with a fragment thereof,under conditions which are suitable for complex formation; and c)detecting complex formation, wherein the presence of such a complexidentifies a small molecule, which binds to the QPCTL.

One method for identifying an antagonist comprises delivering a smallmolecule which binds QPCTL into extracts from cells transformed with avector expressing QPCTL along with a chromogenic substrate (e.g.Ala-Pro-AFC or Ala-Pro-AMC) under conditions where cleavage wouldnormally occur, and then assaying for inhibition of cleavage by theenzyme by monitoring changes in fluorescence, or UV light absorption, byspectrophotometry to identify molecules that inhibit cleavage. A reducedrate of reaction or total amount of fluorescence or UV light absorption,in the presence of the molecule, establishes that the small molecule isan antagonist, which reduces QPCTL catalytic/enzymatic activity. Oncesuch molecules are identified, they may be administered to reduce orinhibit cyclisation of N-terminal Glu- or Gln-residues by a QPCTL.

In accordance with still another specific aspect, the invention providesa method of screening for a compound capable of inhibiting the enzymaticactivity of at least one mature protein according to the presentinvention, preferably selected from the proteins of SEQ ID NOS: 11 to18, which method comprises incubating said mature protein and a suitablesubstrate for said mature protein in the presence of one or more testcompounds or salts thereof, measuring the enzymatic activity of saidmature protein, comparing said activity with comparable activitydetermined in the absence of a test compound, and selecting the testcompound or compounds that reduce the enzymatic activity.

Furthermore, the invention also provides a method of screening for aselective QC-inhibitor, i.e. a compound capable of inhibiting theenzymatic activity of QC, wherein said QC is preferably the protein ofSEQ ID NO: 10, that does not inhibit the enzymatic activity of at leastone mature protein according to the present invention, preferablyselected from the proteins of SEQ ID NOS: 11 to 18, which methodcomprises incubating said mature protein and a suitable substrate in thepresence of one or more inhibitors or salts thereof of QC, measuring theenzymatic activity of said mature protein, comparing said activity withcomparable activity determined in the absence of the QC inhibitor, andselecting a compound that does not reduce the enzymatic activity of saidmature protein.

Furthermore, the invention also provides a method of screening for aselective QPCTL-inhibitor, i.e. a compound capable of inhibiting theenzymatic activity of at least one QPCTL protein, which is preferablyselected from the proteins of SEQ ID NOS: 11 to 18; that does notinhibit the enzymatic activity of QC, wherein said QC is preferably theprotein of SEQ ID NO: 10, which method comprises incubating said QC inthe presence of one or more inhibitors or salts thereof of a QPCTL,measuring the enzymatic activity of QC, comparing said activity withcomparable activity determined in the absence of the QPCTL inhibitor,and selecting a compound that does not reduce the enzymatic activity ofsaid QPCTL protein.

Useful inhibitors of QC, which also could be useful as inhibitors ofQPCTLs, are described in WO 2004/098625, WO 2004/098591, WO 2005/039548and WO 2005/075436, which are incorporated herein in their entirety,especially with regard to the structure of the inhibitors and theirproduction.

Examples of QPCTL-Inhibitors

Potential QPCTL-inhibitors, which are suitable for uses and methodsaccording to the present invention are disclosed in WO 2005/075436,which is incorporated herein in its entirety with regard to thestructure, synthesis and methods of use of the QC-inhibitors.

In particular:

A suitable compound is that of formula 1*:

In a further embodiment, the inhibitors of QPCTL may be those of formula1a,

wherein R is defined in examples 1 to 53.

ESI-MS Example R (M + H) 1 Methyl 199.3 2 tert-Butyl 241.4 3 Benzyl275.4 4 Phenyl 261.4 5 4-(fluoro)-phenyl 279.35 6 4-(chloro)-phenyl295.80 7 4-(ethyl)-phenyl 289.41 8 4-(trifluoromethyl)-phenyl 329.4 94-(methoxy-carbonyl)-Phenyl 319.4 10 4-(acetyl)-phenyl 303.4 114-(methoxy)-phenyl 291.4 12 bicyclo[2.2.1]hept-5-en-2-yl 277.5 133,4-(dimethoxy)-phenyl 321.5 14 2,4-(dimethoxy)-phenyl 321.5 153,5-(dimethoxy)-phenyl 321.5 16 2-(methoxy-carbonyl)-Phenyl 319.4 174-(oxazol-5-y)-phenyl 328.5 18 4-(pyrazol-1-yl)-phenyl 327.4 194-(isopropyl)-phenyl 303.5 20 4-(piperidine-1-sulfonyl)-Phenyl 408.6 214-(morpholin-4-yl)-phenyl 346.5 22 4-(cyano)-phenyl 286.4 232,3-dihydro-benzo[1,4] 319.4 dioxin-6-yl 24 benzo[1,3]dioxol-5-yl 305.425 3,4,5(trimethoxy)-phenyl 351.5 26 3-(methoxy)-phenyl 291.4 274-(ethoxy)-phenyl 305.5 28 4-(benzyloxy)-phenyl 367.5 294-(methoxy)-benzyl 305.5 30 3,4-(dimethoxy)-benzyl 335.5 312-(methoxy-carbonyl)- 325.5 thiophene-3-yl 323-(ethoxy-carbonyl)-4,5,6,7- 392.6 tetrahydrobenzo[b]thio- phene2-yl 332-(methoxy-carbonyl)-4- 339.5 (methyl)-thiophene-3-yl 34Benzo[c][1,2,5]thiazol-4-yl 319.5 35 Benzo[c][1,2,5]thiazol-5-yl 319.536 5-(methyl)-3-(phenyl)- 342.5 isooxazol-4-yl 373,5-(dimethyl)-isooxazol-4-yl 280.4 38 4-(iodo)-phenyl 387.3 394-(bromo)-phenyl 340.3 40 4-(methyl)-phenyl 275.4 41 Naphthalen-1-yl311.5 42 4-(nitro)-phenyl 306.4 43 Butyl 241.4 44 Cyclooctyl 295.5 45Furan-2-ylmethyl 265.4 46 Tetrahydrofuran-2-ylmethyl 269.4 47Benzo[1,3]dioxol-5-ylmethyl 319.4 48 2-(morpholin-4-yl)-ethyl 298.5 494-(methylsulfanyl)-phenyl 307.5 50 4-(dimethylamino)-phenyl 304.5 514-(trifluoromethoxy)-phenyl 345.4 52 Benzoyl 288.3 53 Pyridin-4-yl 261.1

Further suitable inhibitors of QPCTL may be those of formula 1b,

wherein R¹ and R² are defined in examples 54 to 95.

Example R¹ R² 54 Cyano Methyl 55 Cyano 3,4-(dimethoxy)-phenyl 56 Cyano2,4-(dimethoxy)-phenyl 57 Cyano 3,5-(dimethoxy)-phenyl 58 Cyano2,3-dihydrobenzo[b][1,4]dioxin-7-yl 59 Cyano Benzo[d][1,3]dioxol-6-yl 60Cyano 3,4,5-(trimethoxy)-phenyl 61 Cyano 3-(methoxy)-phenyl 62 Cyano4-(ethoxy)-phenyl 63 Cyano 4-(benzyloxy)-phenyl 64 Cyano Phenyl 65 Cyano4-(methoxy)-phenyl 66 Cyano 4-(acetyl)-phenyl 67 Cyano 4-(nitro)-phenyl68 Cyano Benzyl 69 Cyano Naphthalen-1-yl 70 Cyano 4-(fluoro)-phenyl 71Cyano 4-(iodo)-phenyl 72 Cyano 4-(bromo)-phenyl 73 Cyano Cyclooctyl 74Cyano tert-butyl 75 Cyano 4-(methyl)-phenyl 76 Cyano4-(methylthio)-phenyl 77 Cyano 4-(ethyl)-phenyl 78 Cyano4-(dimethylamino)-phenyl 79 Cyano Butyl 80 Cyano Trityl 81 Cyano(Benzo[d][1,3]dioxol-6yl)methyl 82 Cyano (tetrahydrofuran-2yl)methyl 83Cyano 4-(trifluoromethyl)-phenyl 84 Cyano (furan-2-yl)methyl 85 Cyano2-(morpholin-4-yl)-ethyl 86 Cyano 4-(oxazol-5yl)-phenyl 87 CyanoPyridin-3-yl 88 Cyano 4-(cyano)-phenyl 89 Cyano4-(trifluoromethoxy)-phenyl 90 Cyano 4-(piperidinosulfonyl)-phenyl 91Cyano 4-(1H-pyrazol-1-yl)phenyl 92 H 3,4-(dimethoxy)-phenyl 93 Methyl3,4-(dimethoxy)-phenyl 94 Cyano 2,3,4-(trimethoxy)-phenyl 95 CyanoCycloheptyl

Further suitable inhibitors of QPCTL may be those of formula 1c,

wherein R³ is defined in examples 96 to 102.

ESI-MS Example R³ (M + H) 96 Ethyl 197.3 97 6-fluoro-4H-benzo[d] 321.4[1,3]dioxin-8-yl 98 3-(cylopentyloxy)-4- 359.4 (methoxy)-phenyl 994-(heptyloxy)-phenyl 359.5 100 3,4-dihydro-2H-benzo[b] 317.4[1,4]dioxepin-7-yl 101 4-(butoxy)-phenyl 317.4 1023,4-(dimethoxy)-phenyl 305.4

Further suitable inhibitors of QPCTL may be those of formula 1d,

wherein the position on the ring is defined in examples 103 to 105.

Position of the ESI-MS Example Benzyl-substitution (M + H) 103 2 383.5104 3 383.5 105 4 383.5

Further suitable inhibitors of QPCTL may be those of formula 1e,

wherein R⁴ and R⁵ are defined in examples 106 to 109.

ESI-MS Example R⁴ R⁵ (M + H) 106(S) H Methyl 335.5 107(R) Methyl H 335.5108   Methyl Methyl 349.5 109   —CH₂—CH₂— 347.5

Further suitable inhibitors of QPCTL may be those of formula 1f,

wherein R⁶ is defined in examples 110 to 112.

ESI-MS Example R⁶ (M + H) 110 H 259.4 111 Chloro 293.8 112 Methoxy 289.4

Further suitable inhibitors of QPCTL may be those of formula 1g,

wherein R⁷, R⁸ and R⁹ are defined in examples 113 to 132.

ESI-MS Example R⁷ R⁸ R⁹ (M + H) 113 Phenyl H H 260.4 114 Thiophen-2-yl HH 266.5   115(R) Phenyl Methyl H 274.5   116(S) Phenyl H Methyl 274.5117 Phenyl H Ethyl 288.5 118 Phenyl H Phenyl 336.5 119 3,4-(dimethoxy)-H H 320.5 Phenyl 120 3,4-(dimethoxy)- Methyl Methyl 347.2 Phenyl 1214-(chloro)-phenyl —CH₂—CH₂—CH₂— 334.9 122 4-(chloro)-phenyl—CH₂—C₂H₄—CH₂— 349.0 123 4-(methoxy)-phenyl —CH₂—C₃H₆—CH₂— 358.6 1244-(methoxy)-phenyl —CH₂—CH₂— 316.5 125 3,4-(dimethoxy)- —CH₂—CH₂— 346.5Phenyl 126 3,4,5-(trimethoxy)- —CH₂—CH₂— 376.6 Phenyl 1272,3,4-(trimethoxy)- —CH₂—CH₂— 376.6 Phenyl 128 2-(methoxy)-phenyl—CH₂—CH₂— 316.5 129 3-(methoxy)-phenyl —CH₂—CH₂— 316.5 1302,3-(dimethoxy)- —CH₂—CH₂— 346.5 Phenyl 131 3,5-(dimethoxy)- —CH₂—CH₂—346.5 Phenyl 132 2,5-(dimethoxy)- —CH₂—CH₂— 346.5 Phenyl

Further suitable inhibitors of QPCTL may be are those of formula 1h,

wherein n is defined in examples 133 to 135.

ESI-MS Example N (M + H) 133 3 306.4 134 4 320.5 135 5 334.5

Further suitable inhibitors of QPCTL may be those of formula 1i,

wherein m is defined in examples 136 and 137.

ESI-MS Example m (M + H) 136 2 307.4 137 4 335.5

Further suitable inhibitors of QPCTL may be those of formula 138 to 141.

ESI-MS Example Structure (M + H) 138

347.5 139

347.2 140

226.3 141

370.4

The term “agonist”, as used herein, refers to a molecule which, whenbound to QPCTL, increases or prolongs the duration of the effect ofQPCTL. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules that bind to and modulate the effect of QPCTL.Although it is less likely that small molecules will prove to beeffective QPCTL agonists, a method for identifying such a smallmolecule, which binds QPCTL as an agonist, comprises delivering achromogenic form of a small molecule that binds QPCTL into cellstransformed with a vector expressing QPCTL and assaying for fluorescenceor UV light absorption changes by spectrophotometry. An increased amountof UV absorption or fluorescence would establish that the small moleculeis an agonist that increases QPCTL activity.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in published PCT application WO84/03564. In this method, large numbers of different small testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. The test compounds are reacted with QPCTL, or withfragments thereof, and then washed. Bound QPCTL is then detected bymethods well known in the art. Purified QPCTL can also be coateddirectly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding QPCTL specificallycompete with a test compound for binding QPCTL. In this manner,antibodies can be used to detect the presence of any peptide that sharesone or more antigenic determinants with QPCTL.

As indicated above, by investigating the binding sites, ligands may bedesigned that, for example, have more interactions with QPCTL than doits natural ligands. Such antagonist ligands will bind to QPCTL withhigher affinity and so function as competitive ligands. Alternatively,synthetic or recombinant proteins homologous or analogous to the ligandbinding site of native QPCTL may be designed, as may other moleculeshaving high affinity for QPCTL. Such molecules should also be capable ofdisplacing QPCTL and provide a protective effect.

As indicated above, the knowledge of the structures of QPCTL enablessynthetic binding site homologues and analogues to be designed. Suchmolecules will facilitate greatly the use of the binding properties totarget potential therapeutic agents, and they may also be used to screenpotential therapeutic agents. Furthermore, they may be used asimmunogens in the production of monoclonal antibodies, which antibodiesmay themselves be used in diagnosis and/or therapy as describedhereinbefore.

Therapeutic Applications

It is known in the art that amyloid peptides, e.g. Abeta 1-42 (SEQ ID NO23) and Abeta 1-40 (SEQ ID NO 24) become N-terminally truncated byproteolytic enzymes such as for example aminopeptidases or dipeptidylaminopeptidases, resulting in the Abeta-peptides 3-42 (SEQ ID NO 25),3-40 (SEQ ID NO 26), 11-42 (SEQ ID NO 27) and 11-40 (SEQ ID NO 28).These truncated Abeta peptides start with a glutamate residue at theN-terminus and are thus substrates for QC (see also WO 2004/09862) andpossibly also for the QPCTLs of SEQ ID NOS 11-18, 21 and 22, preferablythe human isoQCs of SEQ ID NOS 11, 12, 21 and 22, most preferably thehuman isoQCs of SEQ ID NOS 11 and 12. The resulting pGlu-Abeta peptidesof SED ID NOS 29-32 are much more hydrophobic than the non-pyroglutamtedpeptides, are much more prone to form A-beta peptide aggregates, such asoligomers and fibrills, and were shown to by highly neurotoxic. Finally,the Abeta-peptides of SEQ ID NOS 29-32 play a crucial role in thedevelopment of Alzheimer's disease and Down Syndrome.

Accordingly, inhibitors of the QPCTLs of SEQ ID NOS 11-18, 21 and 22,preferably the human isoQCs of SEQ ID NOS 11, 12, 21 and 22, mostpreferably the human isoQCs of SEQ ID NOS 11 and 12, may be used for thetreatment of amyloid peptide related diseases, especiallyneurodegenerative diseases, in particular Alzheimer's disease and DownSyndrome.

Other potential physiological substrates of QPTCLs in mammals areselected from the group consisting of Glu¹-ABri (SEQ ID NO 33),Glu¹-ADan (SEQ ID NO 34), and Gln¹-Gastrins (17 and 34) (SEQ ID NOS 35and 36). Their pyroglutamated forms (SEQ ID NOS 37-40) cause pathologiessuch as those selected from the group consisting of duodenal cancer withor w/o Helicobacter pylori infections, colorectal cancer,Zolliger-Ellison syndrome, Familial British Dementia (FBD) and FamilialDanish Dementia (FDD). Accordingly, inhibitors of QPCTLs can be used totreat these pathologies.

Further potential physiological substrates of QPCTLs are shown in table3.

TABLE 3 Amino acid sequences of physiological active peptides with an N-terminal glutamine residue Peptide Amino acid sequence FunctionGastrin 17 QGPWL EEEEEAYGWM DF Gastrin stimulates the (SEQ ID NO 35)(amide) stomach mucosa to produce Swiss-Prot: P01350and secrete hydrochloric acid and the pancreas to secrete its digestiveenzymes. It also stimulates smooth muscle contractionand increases blood circulation and water secretion in the stomach andintestine. Neurotensin QLYENKPRRP YIL Neurotensin plays an(SEQ ID NO 41) endocrine or paracrine role Swiss-Prot: P30990in the regulation of fat metabolism. It causes contraction of smoothmuscle. FPP QEP amide A tripeptide related to thyrotrophin releasinghormone (TRH), is found in seminal plasma. Recentevidence obtained in vitro and in vivo showed that FPPplays an important role in regulating sperm fertility. TRH QHP amideTRH functions as a regulator Swiss-Prot: P20396of the biosynthesis of TSH in the anterior pituitary glandand as a neurotransmitter/ neuromodulator in the central and peripheralnervous systems. GnRH QHWSYGL RP(G) amide Stimulates the secretion of(SEQ ID NO 42) gonadotropins; it stimulates Swiss-Prot: P01148the secretion of both luteinizing and follicle- stimulating hormones.CCL16 (small QPKVPEW VNTPSTCCLK Shows chemotactic activityinducible cytokine YYEKVLPRRL VVGYRKALNC for lymphocytes and A16)HLPAIIFVTK RNREVCTNPN monocytes but not (SEQ ID NO 43)DDWVQEYIKD PNLPLLPTRN neutrophils. Also shows Swiss-Prot:LSTVKIITAK NGQPQLLNSQ potent myelosuppressive O15467activity, suppresses proliferation of myeloid progenitor cells.Recombinant SCYA16 shows chemotactic activity for monocytes and THP-1monocytes, but not for resting lymphocytes and neutrophils. Induces acalcium flux in THP-1 cells that were desensitized byprior expression to RANTES. CCL8 (small QPDSVSI PITCCFNVINChemotactic factor that inducible cytokine RKIPIQRLES YTRITNIQCPattracts monocytes, A8) KEAVIFKTKR GKEVCADPKE lymphocytes, basophils and(SEQ ID NO 44) RWVRDSMKHL DQIFQNLKP eosinophils. May play a roleSwiss-Prot: P80075 in neoplasia and inflammatory hostresponses. This protein can bind heparin. CCL2 (small QPDAINA PVTCCYNFTNChemotactic factor that inducible cytokine RKISVQRLAS YRRITSSKCPattracts monocytes and A2) KEAVIFKTIV AKEICADPKQbasophils but not neutrophils (SEQ ID NO 45) KWVQDSMDHL DKQTQTPKTor eosinophils. Augments Swiss-Prot: P13500monocyte anti-tumor activity. Has been implicated in thepathogenesis of diseases characterized by monocyticinfiltrates, like psoriasis, rheumatoid arthritis oratherosclerosis. May be involved in the recruitment ofmonocytes into the arterial wall during the diseaseprocess of atherosclerosis. Binds to CCR2 and CCR4. CCL18 (smallQVGTNKELC CLVYTSWQIP Chemotactic factor that inducible cytokineQKFIVDYSET SPQCPKPGVI attracts lymphocytes but not A18)LLTKRGRQIC ADPNKKWVQK monocytes or granulocytes. (SEQ ID NO 46)YISDLKLNA May be involved in B cell Swiss-Prot: P55774migration into B cell follicles in lymph nodes. Attractsnaive T lymphocytes toward dendritic cells and activatedmacrophages in lymph nodes, has chemotactic activity for naive T cells,CD4+ and CD8+ T cells and thus may play a role in bothhumoral and cell-mediated immunity responses. FractalkineQHHGVT KCNITCSKMT The soluble form is (neurotactin)SKIPVALLIH YQQNQASCGK chemotactic for T cells and (SEQ ID NO 47)RAIILETRQH RLFCADPKEQ monocytes, but not for Swiss-Prot: P78423WVKDAMQHLD RQAAALTRNG neutrophils. The membrane- GTFEKQIGEV KPRTTPAAGGbound form promotes MDESVVLEPE ATGESSSLEP adhesion of those leukocytesTPSSQEAQRA LGTSPELPTG to endothelial cells. May playVTGSSGTRLP PTPKAQDGGP a role in regulating leukocyteVGTELFRVPP VSTAATWQSS adhesion and migration APHQPGPSLW AEAKTSEAPSprocesses at the TQDPSTQAST ASSPAPEENA endothelium. Binds toPSEGQRVWGQ GQSPRPENSL cx3cr1. EREEMGPVPA HTDAFQDWGPGSMAHVSVVP VSSEGTPSRE PVASGSWTPK AEEPIHATMD PQRLGVLITP VPDAQAATRRQAVGLLAFLG LLFCLGVAMF TYQSLQGCPR KMAGEMAEGL RYIPRSCGSN SYVLVPVCCL7 (small QPVGINT STTCCYRFIN Chemotactic factor thatinducible cytokine KKIPKQRLES YRRTTSSHCP attracts monocytes and A7)REAVIFKTKL DKEICADPTQ eosinophils, but not (SEQ ID NO 48)KWVQDFMKHL DKKTQTPKL neutrophils. Augments Swiss-Prot: P80098monocyte anti-tumor activity. Also induces the release ofgelatinase B. This protein can bind heparin. Binds toCCR1, CCR2 and CCR3. Orexin A QPLPDCCRQK TCSCRLYELLNeuropeptide that plays a (Hypocretin-1) HGAGNHAAGI LTLsignificant role in the (SEQ ID NO 49) regulation of food intake andSwiss-Prot O43612 sleep-wakefulness, possiblyby coordinating the complex behavioral and physiologicresponses of these complementary homeostatic functions. It plays also abroader role in the homeostatic regulation of energy metabolism,autonomic function, hormonal balance and the regulation of body fluids.Orexin-A binds to both OX1R and OX2R with a high 7affinity. Substance PRPK PQQFFGLM Belongs to the tachykinins. (SEQ ID NO 50)Tachykinins are active peptides which excite neurons, evoke behavioralresponses, are potent vasodilators and secretagogues, and contract(directly or indirectly) many smooth muscles.

The peptides Gln¹-Gastrin (17 and 34 amino acids in length),Gln¹-Neurotensin and Gln¹-FPP were identified as new physiologicalsubstrates of QPCTLs. Gastrin, Neurotensin and FPP comprise a pGluresidue in their N-terminal position. This N-terminal pGlu residue maybe formed from N-terminal glutamine by QPCTL catalysis for all peptides.As a result, these peptides are activated in terms of their biologicalfunction upon conversion of the N-terminal glutamine residue to pGlu.

Transepithelial transducing cells, particularly the gastrin (G) cell,co-ordinate gastric acid secretion with the arrival of food in thestomach. Recent work showed that multiple active products are generatedfrom the gastrin precursor, and that there are multiple control pointsin gastrin biosynthesis. Biosynthetic precursors and intermediates(progastrin and Gly-gastrins) are putative growth factors; theirproducts, the amidated gastrins, regulate epithelial cell proliferation,the differentiation of acid-producing parietal cells andhistamine-secreting enterochromaffin-like (ECL) cells, and theexpression of genes associated with histamine synthesis and storage inECL cells, as well as acutely stimulating acid secretion. Gastrin alsostimulates the production of members of the epidermal growth factor(EGF) family, which in turn inhibit parietal cell function but stimulatethe growth of surface epithelial cells. Plasma gastrin concentrationsare elevated in subjects with Helicobacter pylori, who are known to haveincreased risk of duodenal ulcer disease and gastric cancer (Dockray, G.J. 1999 J Physiol 15 315-324).

The peptide hormone gastrin, released from antral G cells, is known tostimulate the synthesis and release of histamine from ECL cells in theoxyntic mucosa via CCK-2 receptors. The mobilized histamine induces acidsecretion by binding to the H(2) receptors located on parietal cells.Recent studies suggest that gastrin, in both its fully amidated and lessprocessed forms (progastrin and glycine-extended gastrin), is also agrowth factor for the gastrointestinal tract. It has been establishedthat the major trophic effect of amidated gastrin is for the oxynticmucosa of stomach, where it causes increased proliferation of gastricstem cells and ECL cells, resulting in increased parietal and ECL cellmass. On the other hand, the major trophic target of the less processedgastrin (e.g. glycine-extended gastrin) appears to be the colonic mucosa(Koh, T. J. and Chen, D. 2000 Regul Pept 9337-44).

In a further embodiment, the present invention provides the use ofactivity increasing effectors of QPCTLs for the stimulation ofgastrointestinal tract cell proliferation, especially gastric mucosalcell proliferation, epithelial cell proliferation, the differentiationof acid-producing parietal cells and histamine-secretingenterochromaffin-like (ECL) cells, and the expression of genesassociated with histamine synthesis and storage in ECL cells, as well asfor the stimulation of acute acid secretion in mammals by maintaining orincreasing the concentration of active pGlu¹-Gastrin (SEQ ID NOS 39 and40).

In a further embodiment, the present invention provides the use ofinhibitors of QPCTLs for the treatment of duodenal ulcer disease andgastric cancer with or w/o Helicobacter pylori infections in mammals bydecreasing the conversion rate of inactive Gln¹-Gastrin (SEQ ID NOS 35and 36) to active pGlu¹-Gastrin (SEQ ID NOS 39 and 40).

Neurotensin (NT) (SEQ ID NO 41) is a neuropeptide implicated in thepathophysiology of schizophrenia that specifically modulatesneurotransmitter systems previously demonstrated to be misregulated inthis disorder. Clinical studies in which cerebrospinal fluid (CSF) NTconcentrations have been measured revealed a subset of schizophrenicpatients with decreased CSF NT concentrations that are restored byeffective antipsychotic drug treatment. Considerable evidence alsoexists concordant with the involvement of NT systems in the mechanism ofaction of antipsychotic drugs. The behavioral and biochemical effects ofcentrally administered NT remarkably resemble those of systemicallyadministered antipsychotic drugs, and antipsychotic drugs increase NTneurotransmission. This concatenation of findings led to the hypothesisthat NT functions as an endogenous antipsychotic. Moreover, typical andatypical antipsychotic drugs differentially alter NT neurotransmissionin nigrostriatal and mesolimbic dopamine terminal regions, and theseeffects are predictive of side effect liability and efficacy,respectively (Binder, E. B. et al. 2001 Biol Psychiatry 50 856-872).

Accordingly, the present invention provides the use of activityincreasing effectors of QPCTLs for the preparation of antipsychoticdrugs and/or for the treatment of schizophrenia in mammals. Theeffectors of QPCTLs either maintain or increase the concentration ofactive pGlu¹-neurotensin.

Fertilization promoting peptide (FPP), a tripeptide related tothyrotrophin releasing hormone (TRH), is found in seminal plasma. Recentevidence obtained in vitro and in vivo showed that FPP plays animportant role in regulating sperm fertility. Specifically, FPPinitially stimulates nonfertilizing (uncapacitated) spermatozoa to“switch on” and become fertile more quickly, but then arrestscapacitation so that spermatozoa do not undergo spontaneous acrosomeloss and therefore do not lose fertilizing potential. These responsesare mimicked, and indeed augmented, by adenosine, known to regulate theadenylyl cyclase (AC)/cAMP signal transduction pathway. Both FPP andadenosine have been shown to stimulate cAMP production in uncapacitatedcells but inhibit it in capacitated cells, with FPP receptors somehowinteracting with adenosine receptors and G proteins to achieveregulation of AC. These events affect the tyrosine phosphorylation stateof various proteins, some being important in the initial “switching on,”others possibly being involved in the acrosome reaction itself.Calcitonin and angiotensin II, also found in seminal plasma, havesimilar effects in vitro on uncapacitated spermatozoa and can augmentresponses to FPP. These molecules have similar effects in vivo,affecting fertility by stimulating and then maintaining fertilizingpotential. Either reductions in the availability of FPP, adenosine,calcitonin, and angiotensin II or defects in their receptors contributeto male infertility (Fraser, L. R. and Adeoya-Osiguwa, S. A. 2001 VitamHorm 63, 1-28).

In a further embodiment, the present invention provides the use ofinhibitors of QPCTLs for the preparation of fertilization prohibitivedrugs and/or to reduce the fertility in mammals. The inhibitors ofQPCTLs decrease the concentration of active pGlu¹-FPP, leading to aprevention of sperm capacitation and deactivation of sperm cells. Incontrast it could be shown that activity increasing effectors of QC areable to stimulate fertility in males and to treat infertility.

In a further embodiment, further physiological substrates of QPCTLs wereidentified within the present invention. These are Gln¹-CCL2 (SEQ ID NO45), Gln¹-CCL7 (SEQ ID NO 48), Gln¹-CCL8 (SEQ ID NO 44), Gln¹-CCL16 (SEQID NO 43), Gln¹-CCL18 (SEQ ID NO 46) and Gln¹-fractalkine (SEQ ID NO47). For details see Table 3. These polypeptides play an important rolein pathophysiological conditions, such as suppression of proliferationof myeloid progenitor cells, neoplasia, inflammatory host responses,cancer, psoriasis, rheumatoid arthritis, atherosclerosis, humoral andcell-mediated immunity responses, leukocyte adhesion and migrationprocesses at the endothelium.

Several cytotoxic T lymphocyte peptide-based vaccines against hepatitisB, human immunodeficiency virus and melanoma were recently studied inclinical trials. One interesting melanoma vaccine candidate alone or incombination with other tumor antigens, is the decapeptide ELA. Thispeptide is a Melan-A/MART-1 antigen immunodominant peptide analog, withan N-terminal glutamic acid. It has been reported that the amino groupand gamma-carboxylic group of glutamic acids, as well as the amino groupand gamma-carboxamide group of glutamines, condense easily to formpyroglutamic derivatives. To overcome this stability problem, severalpeptides of pharmaceutical interest have been developed with apyroglutamic acid instead of N-terminal glutamine or glutamic acid,without loss of pharmacological properties. Unfortunately compared withELA, the pyroglutamic acid derivative (PyrELA) and also the N-terminalacetyl-capped derivative (AcELA) failed to elicit cytotoxic T lymphocyte(CTL) activity. Despite the apparent minor modifications introduced inPyrELA and AcELA, these two derivatives probably have lower affinitythan ELA for the specific class I major histocompatibility complex.Consequently, in order to conserve full activity of ELA, the formationof PyrELA must be avoided (Beck A. et al. 2001, J Pept Res57(6):528-38.). Recently, it was found that also the enzyme glutaminylcyclase (QC) is overexpressed in melanomas (Ross D. T et al., 2000, NatGenet. 24:227-35.).

Accordingly, the present invention provides the use of inhibitors ofQPCTLs for the preparation of a medicament for the treatment ofpathophysiological conditions, such as suppression of proliferation ofmyeloid progenitor cells, neoplasia, inflammatory host responses,cancer, malign metastasis, melanoma, psoriasis, rheumatoid arthritis,atherosclerosis, impaired humoral and cell-mediated immunity responses,leukocyte adhesion and migration processes at the endothelium.

Furthermore, Gln¹-orexin A (SEQ ID NO 49) was identified as aphysiological substrate of QPCTLs within the present invention. Orexin Ais a neuropeptide that plays a significant role in the regulation offood intake and sleep-wakefulness, possibly by coordinating the complexbehavioral and physiologic responses of these complementary homeostaticfunctions. It plays also a role in the homeostatic regulation of energymetabolism, autonomic function, hormonal balance and the regulation ofbody fluids.

In a further embodiment, the present invention provides the use ofinhibitors of QPCTLs for the preparation of a medicament for thetreatment of impaired food intake and sleep-wakefulness, impairedhomeostatic regulation of energy metabolism, impaired autonomicfunction, impaired hormonal balance and impaired regulation of bodyfluids.

Polyglutamine expansions in several proteins lead to neurodegenerativedisorders, such as Parkinson disease and Kennedy's disease. Themechanism therefore remains largely unknown. The biochemical propertiesof polyglutamine repeats suggest one possible explanation: endolyticcleavage at a glutaminyl-glutaminyl bond followed by pyroglutamateformation may contribute to the pathogenesis through augmenting thecatabolic stability, hydrophobicity, amyloidogenicity, and neurotoxicityof the polyglutaminyl proteins (Saido, T; Med Hypotheses (2000) Mar;54(3):427-9). Accordingly, the present invention provides therefore theuse of inhibitors of QPCTLs for the preparation of a medicament for thetreatment of Parkinson disease and Huntington's disease.

A further substrate of QPTCLs is the peptide QYNAD (SEQ ID NO 51). Itspyroglutamated form pGlu-Tyr-Asn-Ala-Asp (pEYNAD) (SEQ ID NO 52) is theeffective agent with blocking activity of voltage-gated sodium channels.Sodium channels are expressed at high density in myelinated axons andplay an obligatory role in conducting action potentials along axonswithin the mammalian brain and spinal cord. Therefore, it is speculatedthat they are involved in several aspects of the pathophysiology ofmultiple sclerosis (MS), the Guillain-Barré syndrome and chronicinflammatory demyelinizing polyradiculoneuropathy.

In a further embodiment, the present invention provides the use ofinhibitors of QPCTLs for the preparation of a medicament for thetreatment of inflammatory autoimmune diseases, especially for multiplesclerosis, the Guillain-Barré syndrome and chronic inflammatorydemyelinizing polyradiculoneuropathy, wherein the formation of thevoltage-gated sodium channel blocking peptide pEYNAD is inhibited.

Furthermore, the present invention provides a diagnostic assay,comprising a QC-inhibitor.

In another embodiment, the present invention provides a method ofdiagnosing any one of the aforementioned diseases and/or conditions,comprising the steps of

-   -   collecting a sample from a subject who is suspected to be        afflicted with said disease and/or condition,    -   contacting said sample with a QC-inhibitor, and    -   determining whether or not said subject is afflicted by said        disease and/or condition.

Preferably, the sample in said diagnosing method is a blood sample, aserum sample, a sample of cerebrospinal liquor or a urine sample.

Preferably, the subject in said diagnosing method is a human being.

Preferably, the QC inhibitor in said diagnosing method is a selective QCinhibitor.

Further preferred for the use in said diganostic assay are selectiveQPCTL inhibitors.

The present invention further pertains to a diagnostic kit for carryingout the dignosing method comprising as detection means theaforementioned diagnostic assay and a determination means.

Example 1 Preparation of Human isoQC Cell Lines and Media

African green monkey kidney cell line COS-7, human neuroblastoma cellline SH-SY5Y, human asatrocytoma cell line LN405, human keratinocytomacell line HaCaT and human hepatocellular carcinoma cell line Hep-G2 werecultured in appropriate cell culture media (DMEM, 10% FBS for Cos-7,SH-SY5Y, LN405, HaCaT), (RPMI1640, 10% FBS for Hep-G2), in a humidifiedatmosphere of 5% CO₂ (HaCaT, Hep-G2, COS-7) or 10% CO₂ (SH-SY5Y, LN405)at 37° C.

Analysis of Human isoQC Expression Using RT-PCR

Total RNA was isolated from SH-SY5Y, LN405, HaCaT and Hep-G2 cells usingthe RNeasy Mini Kit (Qiagen) and reversely transcribed by SuperScript II(Invitrogen). Subsequently, human isoQC was amplified on a 1:12.5dilution of generated cDNA product in a 25 μl reaction with HerculaseEnhanced DNA-Polymerase (Stratagene) using primers isoQCh-1 (sense, SEQID NO: 53) and isoQCh-2 (antisense, SEQ ID NO: 54). The PCR product ofHep-G2 was purified utilizing the Strataprep PCR Purification Kit(Stratagene) and confirmed by sequencing.

Results

Analysis of Human isoQC Expression Using RT-PCR

Transcripts of human isoQC were found to be present in cell linesSH-SY5Y (FIG. 6, lane 1), LN405 (FIG. 6, lane 2), HaCaT (FIG. 6, lane 3)and Hep-G2 (FIG. 6, lane 4). The PCR product of Hep-G2 was confirmed bysequencing.

Isolation of Human isoQC

Full-length cDNA of human isoQC was isolated from Hep-G2 cells usingRT-PCR. Briefly, total RNA of Hep-G2 cells was reversely transcribed bySuperScript II (Invitrogen). Subsequently, human isoQC was amplified ona 1:12.5 dilution of generated cDNA product in a 25 μl reaction withHerculase Enhanced DNA-Polymerase (Stratagene) using primers isoQChu-1(sense, SEQ ID NO: 55) and isoQChu-2 (antisense, SEQ ID NO: 56). Theresulting PCR-product was subcloned into vector pPCRScript CAM SK (+)(Stratagene) and confirmed by sequencing.

Example 2 Preparation and Expression of Human isoQC in Mammalian CellCulture

Molecular Cloning of Plasmid Vectors Encoding a Human isoQC-EGFP FusionProtein

All cloning procedures were done applying standard molecular biologytechniques. For expression of human isoQC-EGFP fusion protein in humancells, the vector pEGFP-N3 (Invitrogen) was used. The cDNA of the nativehuman isoQC starting either at methionine I or at methionine II wasfused N-terminally in frame with the plasmid encoded enhanced greenfluorescent protein (EGFP). The primers isoQC EGFP-1 Met I (SEQ ID NO:57) and isoQC EGFP-3 (SEQ ID NO: 59) were used for amplification ofhuman isoQC starting with methionine I and primers isoQC EGFP-2 Met II(SEQ ID NO: 58) and isoQC EGFP-3 (SEQ ID NO: 59) were used foramplification of human isoQC starting with methionine II. The fragmentswere inserted into vector pEGFP-N3 (Invitrogen) employing therestriction sites of EcoRI and Sail and the correct insertion wasconfirmed by sequencing. Subsequently, the vectors were isolated forcell culture purposes using the EndoFree Maxi Kit (Qiagen).

Cloning Procedure of the N-Terminal Sequences of hisoQC

In addition, the EGFP sequence of vector pEGFP-N3 (Invitrogen) wasintroduced into vector pcDNA 3.1 (Invitrogen) using EGFP-1 (sense) (SEQID NO: 85) and EGFP-2 (antisense) (SEQ ID NO: 86) for amplification. Thefragment was introduced into XhoI site of pcDNA 3.1. The N-terminalsequences of hisoQC beginning with methionine I and II each ending atserine 53 were fused C-terminally with EGFP in vector pcDNA 3.1 usingisoQC EGFP-1 Met I (sense, SEQ ID NO: 57) and hisoQC SS EGFP pcDNA as(antisense) (SEQ ID NO: 87) for the N-terminal fragment of hisoQCbeginning with methionine I and isoQC EGFP-2 Met II (sense, SEQ ID NO:58) and hisoQC SS EGFP pcDNA as (antisense) (SEQ ID NO: 87) for theN-terminal fragment of hisoQC beginning with methionine II. Fragmentswere inserted into EcoRI and NotI restrictione sites of vector pcDNA3.1. Subsequently, the vectors were isolated for cell culture purposesusing the EndoFree Maxi Kit (Qiagen).

Cloning Procedure for Native Expression of hisoQC and hQC

Native hQC was inserted into HindIII and NotI restriction sites andnative hisoQC was inserted into EcoRI and NotI restriction sites ofvector pcDNA 3.1 (+) (Invitrogen) after amplification utilizing primershQC-1 (sense) (SEQ ID NO: 82) and hQC-2 (antisense) (SEQ ID NO: 83) forhQC, isoQC EGFP-1 Met I (sense) (SEQ ID NO: 57) and hisoQC pcDNA as(antisense) (SEQ ID NO: 84) for hisoQC starting with methionine I andisoQC EGFP-2 Met II (sense) (SEQ ID NO: 58) and hisoQC pcDNA as(antisense) (SEQ ID NO: 84) for hisoQC starting with methionine II.

Cloning Procedure for FLAG-Tagged hisoQC and hQC

Human QC was cloned with a C-terminal FLAG-tag after amplificationapplying primers hQC-1 (sense) (SEQ ID NO: 82) and hQC C-FLAG pcDNA as(antisense) (SEQ ID NO: 88) into HindIII and NotI restriction sits ofvector pcDNA 3.1. Human isoQC was inserted with a C-terminal FLAG-taginto pcDNA 3.1 after amplification using primers isoQC EGFP-1 Met I(sense) (SEQ ID NO: 57) and hisoQC C-FLAG pcDNA as (antisense) (SEQ IDNO: 89) for hisoQC starting with methionine 1 and primers isoQC EGFP-2Met II (sense) (SEQ ID NO: 58) and hisoQC C-FLAG pcDNA as (antisense)(SEQ ID NO: 89) for hisoQC starting with methionine 2.

Example 3 Immunhistochemical Staining of Human isoQC in Mammalian CellsTransfection and Histochemical Staining of Cos-7 and LN405

For expression of human isoQC-EGFP fusion proteins starting either withmethionine I or methionine II, COS-7 and LN405 were cultured in 6-welldishes containing a cover slip. Cells were grown until 80% confluency,transfected using Lipofectamin2000 (Invitrogen) according tomanufacturer's manual and incubated in the transfection solution for 5hours. Afterwards, the solution was replaced by appropriate growth mediaand cells were grown over night.

The next day, cells were washed twice with D-PBS (Invitrogen) and fixedusing ice-cold methanol for 10 min at −20° C., followed by 3 washingsteps using D-PBS for 10 min at room temperature. For staining of thegolgi-zone, COS-7 and LN405 were incubated with rabbit anti-mannosidaseII polyclonal antibody (Chemicon) in a 1:50 dilution of antibody inD-PBS for 3 h. For staining of mitochondria in COS-7 and LN405, cellswere incubated with mouse anti-human mitochondria monoclonal antibody(Chemicon) in a 1:100 dilution of antibody in D-PBS for 3 h at roomtemperature. Subsequently, the cells were washed 3 times with D-PBS for10 min. Cells stained for golgi-zone were incubated with goatanti-rabbit IgG secondary antibody conjugated with Rhodamin-RedX(Dianova) for 45 min at room temperature in the dark. Cells stained formitochondria were incubated with goat anti-mouse IgG secondary antibodyconjugated with Rhodamin-RedX (Dianova) for 45 min at room temperaturein the dark. Afterwards, cells were washed 3 times with D-PBS for 5 minat room temperature and at least, the cover slips were mounted on amicroscope slide with citiflour. Cells were observed under afluorescence microscope (Carl-Zeiss).

Results 1. Transfection and Histochemical Staining of LN405

The expression of human isoQC-EGFP fusion protein starting withmethionine I and methionine II in cell line LN405 (green fluorescence)leads to a compartmentalization of the resulting protein.Counterstaining of the golgi-zone of LN405 using mannosidase II antibody(red fluorescence) and subsequent superimposition of human isoQC-EGFPwith mannosidase II suggests a localization of human isoQC-EGFP fusionprotein within the golgi-compartment (yellow coloration of the mergedimages) (FIG. 7,9). Thereby, it is evident that human isoQC starting atmethionine II is sufficient to generate a golgi-localization of thehuman isoQC fusion protein.

The expression of human isoQC-EGFP fusion protein starting withmethionine I and II (green fluorescence) and counterstaining formitochondria (red fluorescence) did not reveal a localization of humanisoQC-EGFP fusion protein starting with methionine I or II within themitochondria due to the absence of a yellow coloration of the mergedimages after superimposition (FIG. 8, 10).

2. Transfection and Histochemical Staining of Cos-7

In analogy to the expression of human isoQC-EGFP fusion protein startingwith methionine I and methionine II in cell line LN405, leads theexpression of human isoQC-EGFP fusion protein starting with methionine Iand methionine II in COS-7 to a compartmentalization of the resultingprotein (green fluorescence). Counterstaining of the golgi-zone of COS-7cells using mannosidase II antibody (red fluorescence) and subsequentsuperimposition of human isoQC-EGFP with mannosidase II suggests alocalization of human isoQC-EGFP fusion protein within thegolgi-compartment of COS-7 (yellow coloration of the merged images)(FIG. 11,13). Again, in COS-7 cells the expression of human isoQC-EGFPfusion protein starting at methionine II is sufficient to cause agolgi-localization.

As expected, the expression of human isoQC-EGFP fusion protein startingwith methionine I and II in COS-7 (green fluorescence) andcounterstaining for mitochondria (red fluorescence) did not result in alocalization of human isoQC-EGFP fusion protein starting with methionineI or II within the mitochondria due to the absence of a yellowcoloration of the merged images after superimposition (FIG. 12,14).

Example 4 Expression and Purification of Human isoQC in E. Coli HostStrains and Media

Escherichia coli strain DH5a was used for propagation of plasmids and E.coli strain BL21 was used for the expression of human isoQC. E. colistrains were grown, transformed and analyzed according to themanufacturer's instructions (Qiagen(DH5α) Stratagene (BL21)). The mediarequired for E. coli, i.e. Luria-Bertani (LB) medium, was preparedaccording to the manufacturer's recommendations.

Molecular Cloning of Plasmid Vectors Encoding the Human QC

All cloning procedures were done applying standard molecular biologytechniques. For expression in E. coli BL21, the vector pET41a (Novagen)was used. The cDNA of the mature human isoQC starting with codon 30(counting from methionine II) was fused in frame with the plasmidencoded GST-tag. After amplification utilizing the primers hisoQCpET41a-1 (SEQ ID NO: 60) and hisoQC pET41a-2 (SEQ ID NO: 61) (Table 4) aN-terminal protease cleavage site for Enterokinase and a C-terminal(His)₆-tag was introduced. After subcloning, the fragment was insertedinto the expression vector employing the restriction sites of Spe I andEcoR I.

Expression and Purification in E. coli BL21

The construct encoding the human isoQC was transformed into BL21 cells(Stratagene) and grown on selective LB agar plates at 37° C. Proteinexpression was carried out in LB medium containing 1% glucose at 37° C.After reaching an OD₆₀₀ of approximately 0.8, isoQC expression wasinduced with 20 μM IPTG for 4 h at 37° C. Cells were separated from themedium by centrigugation (4000×g, 20 min), resuspended in PBS (140 mMNaCl, 2.7 mM KCl, 10 mM Na₂ HPO₄, 1.8 mM KH₂ PO₄, pH 7.3) and lysed byone cycle of freezing and thawing followed by one cycle of French Press.The cell lysate was diluted to a final volume of 1.5 l usingphosphate-containing buffer (50 mM Na₂ HPO₄, 500 mM NaCl. pH 7.3) andcentrifuged at 13.400×g at 4° C. for 1 h. After centrifugation, theprotein concentration of the resulting supernatant was determined usingthe method of Bradford. If necessary, the solution was diluted again toobtain a final total protein concentration of 0.6 mg/ml. The GST-isoQCfusion protein was purified utilizing a 4-step protocol (Table 5). Thepurification is illustrated by SDS-PAGE analysis in FIG. 20.

Example 5 Assays for Glutaminyl Cyclase Activity Fluorometric Assays

All measurements were performed with a NovoStar reader for microplates(BMG Labtechnologies) at 30° C. QC activity was evaluatedfluorometrically using H-Gln-/NA. The samples consisted of 0.2 mMfluorogenic substrate, 0.25 U pyroglutamyl aminopeptidase (Qiagen,Hilden, Germany) in 0.05 M Tris/HCl, pH 8.0 and an appropriately dilutedaliquot of QC in a final volume of 250 μl. Excitation/emissionwavelengths were 320/410 nm. The assay reactions were initiated byaddition of glutaminyl cyclase. QC activity was determined from astandard curve of β-naphthylamine under assay conditions. One unit isdefined as the amount of QC catalyzing the formation of 1 μmol pGlu-βNAfrom H-Gln-βNA per minute under the described conditions.

In a second fluorometric assay, QC was activity was determined usingH-Gln-AMC as substrate. Reactions were carried out at 30° C. utilizingthe NOVOStar reader for microplates (BMG Labtechnologies). The samplesconsisted of varying concentrations of the fluorogenic substrate, 0.1 Upyroglutamyl aminopeptidase (Qiagen) in 0.05 M Tris/HCl, pH 8.0 and anappropriately diluted aliquot of QC in a final volume of 250 μl.Excitation/emission wavelengths were 380/460 nm. The assay reactionswere initiated by addition of glutaminyl cyclase. QC activity wasdetermined from a standard curve of 7-amino-4-methylcoumarin under assayconditions. The kinetic data were evaluated using GraFit software.

Spectrophotometric Assay of isoQC

This assay was used to determine the kinetic parameters for most of theQC substrates. QC activity was analyzed spectrophotometrically using acontinuous method (Schilling, S. et al., 2003 Biol Chem 384, 1583-1592)utilizing glutamic dehydrogenase as auxiliary enzyme. Samples consistedof the respective QC substrate, 0.3 mM NADH, 14 mM α-Ketoglutaric acidand 30 U/ml glutamic dehydrogenase in a final volume of 250 μl.Reactions were started by addition of QC and pursued by monitoring ofthe decrease in absorbance at 340 nm for 8-15 min. The initialvelocities were evaluated and the enzymatic activity was determined froma standard curve of ammonia under assay conditions. All samples weremeasured at 30° C., using the Sunrise reader for microplates. Kineticdata were evaluated using GraFit software.

Inhibitor Assay

For inhibitor testing, the sample composition was the same as describedabove, except of the putative inhibitory compound added. For a rapidtest of QC-inhibition, samples contained 4 mM of the respectiveinhibitor and a substrate concentration at 1 K_(M). For detailedinvestigations of the inhibition and determination of K_(i)-values,influence of the inhibitor on the auxiliary enzymes was investigatedfirst. In every case, there was no influence on either enzyme detected,thus enabling the reliable determination of the QC inhibition. Theinhibitory constant was evaluated by fitting the set of progress curvesto the general equation for competitive inhibition using GraFitsoftware.

Results

A variety of different substrates was evaluated on conversion by humanisoQC (Table 3). All analyzed substrates were converted by isoQC,indicating a relatively relaxed overall specificity similar to human QC(Schilling, S. et al., 2003 Biol Chem 384, 1583-1592). As observedpreviously for human QC (Schilling, S. et al., 2003 Biol Chem 384,1583-1592), highest specificity constants (k_(cat)/K_(M)) were observedfor substrates carrying large hydrophobic amino acids adjacent to theN-terminal glutaminyl residue, e.g. Gln-AMC. In contrast, negativelycharged residues in that very position led to a drastic drop inspecificity, as observed for Gln-Glu, indicating a negatively chargedactive site of isoQC. Compared to human QC, both recombinant iosQCsexerted a lower enzymatic activity (FIG. 21). The difference was up toone order of magnitude. According to the specificity of isoQC, itreasonable to assume that the enzyme is responsible for conversion ofdifferent substrates in vivo, i.e. isoQC is involved in the generationof many different physiological substrates.

Human isoQC activity was competitively inhibited by imidazolederivatives (table 6, FIG. 15). The inhibition constants K_(i) forimidazole and benzimidazole was very similar to the value which wasobtained for human QC previously. A 10-fold drop in K_(i), however, wasobserved for the potent QC inhibitor P150/03. Thus, the binding mode ofthe chelating part, i.e. the imidazole ring, appears to be very similar.Presumably, this results from complexation of the active site zinc ionof QC and isoQC by the imidazole basic nitrogen. The differences in theK_(i)-values for P150/03 clearly demonstrates that the active sites ofboth enzymes display subtle differences. Therefore, it is possible togenerate inhibitors that exert selectivity for one enzymic isoform.Selective inhibitors are beneficial for the treatment of the diseases.

TABLE 3 Kinetic evaluation of peptide substrates of human QC and humanisoQC. Human isoQC was expressed in E. coli BL21 (hisoQCdt) or P.pastoris (YSShisoQC). The substrates are displayed in the one-lettercode of amino acids. k_(cat)/K_(M) k_(cat)/K_(M) K_(M) (mM) K_(M) (mM)k_(cat) (s⁻¹) k_(cat) (s⁻¹) (mM⁻¹ * s⁻¹) (mM⁻¹ * s⁻¹) Substrate hisoQCdtYSShisoQC hisoQCdt YSShisoQC hisoQCdt YSShisoQC Q-βNA 0.03 ± 0.002 0.035± 0.0005 3.37 ± 0.12  8.16 ± 0.87  93.26 ± 6.68 228.70 ± 22.22 QAMC 0.01± 0.0009  0.03 ± 0.0064 1.07 ± 0.03  3.72 ± 0.44  62.57 ± 5.68 102.87 ±29.22 QQ 0.11 ± 0.027  0.11 ± 0.007 2.72 ± 0.25  6.08 ± 0.17  24.50 ±4.009  54.32 ± 4.61 QE  0.7 ± 0.13  0.61 ± 0.064 2.64 ± 0.21  5.33 ±0.43  3.85 ± 0.56  8.75 ± 0.87 QG 0.42 ± 0.04  0.36 ± 0.047 1.65 ± 0.04 3.24 ± 0.18  3.93 ± 0.31  9.01 ± 1.75 QGP 0.21 ± 0.016  0.23 ± 0.024.01 ± 0.14  8.98 ± 0.07  18.82 ± 1.26  38.42 ± 3.55 QYA 0.22 ± 0.01 0.08 ± 0.022  7.7 ± 0.4 16.47 ± 0.72  66.48 ± 13.07  206.9 ± 57.54 QFA0.11 ± 0.016 0.104 ± 0.025 7.49 ± 0.28 11.68 ± 2.39  33.03 ± 2.38 116.99± 34.37 QEYF 0.03 ± 0.004  0.04 ± 0.004 3.34 ± 0.15  5.64 ± 0.39 109.57± 21.03 122.56 ± 5.6 QEDL 0.63 ± 0.052  0.16 ± 0.01 6.41 ± 0.15  9.24 ±0.65  10.2 ± 0.84  55.04 ± 5.14

TABLE 4 Utilized primers Primer Sequence 5′ → 3′ Application IsoQCh-1GGTCTACACCATTTGGAGCGGCTGGC Cell Line (SEQ ID NO: 53) Screening IsoQCh-2GGGTTGGAAGTACATCACTTCCTGGGG Cell Line (SEQ ID NO: 54) ScreeningIsoQChu-1 ACCATGCGTTCCGGGGGCCGCGGG Isolation of (SEQ ID NO: 55) hisoQCIsoQChu-2 ACGCTAGAGCCCCAGGTATTCAGCCAG Isolation of (SEQ ID NO: 56)hisoQC IsoQC EGFP-1 Met ATATATGAATTCATGCGTTCCGGGGGCCGC Cloning human IisoQC (Met I) (SEQ ID NO: 57) into vector pEGFP-N3 IsoQC EGFP-2 MetATATATGAATTCATGGAGCCACTCTTGCCGCCG Cloning human II isoQC (Met II)(SEQ ID NO: 58) into vector pEGFP-N3 IsoQC EGFP-3ATATATGTCGACGAGCCCCAGGTATTCAGCCAG Cloning human (SEQ ID NO: 59)isoQC (Met I and Met II) into vector pEGFP-N3 HisoQC pET41a-1ATATACTAGTGATGACGAC Cloning human (SEQ ID NO: 60)GACAAGTTCTACACCATTTGGAGCG isoQC into vector pET41a HisoQC pET41a-2TATAGAATTCCTAGTGATGGT Cloning human (SEQ ID NO: 61)GATGGTGATGGAGCCCCAGGTATTCAGC isoQC into vector pET41a hisoQC HIS C-TermATA TGA ATT CTT CTA CAC CAT TTG GAG C Cloning human pPICZAA-1 isoQC into(SEQ ID NO: 62) vector PPICZαA hisoQC HIS N-TermATA TGA ATT CCA TCA CCA TCA CCA TCA CTT CTA CAC Cloning human pPICZAA-1CAT TTG GAG CGG C isoQC into (SEQ ID NO: 63) vector PPICZαAhisoQC HIS N-Term 5′- ATA TAT GCG GCC GCC TAG AGC CCC AGG TAT TCACloning human pPICZAA-2 GC-3′ isoQC into (SEQ ID NO: 64) vector PPICZαAisoQCm RT s CCA GGA TCC AGG CTA TTG AG Real-time PCR (SEQ ID NO: 65)analysis of isoQC hisoQC HIS C-TermATA TAT GCG GCC GCC TAG TGA TGG TGA TGG TGA TGG Cloning human pPICZAA-2AGC CCC AGG TAT TCA GCC AG isoQC into (SEQ ID NO: 66) vector PPICZαAisoQCm RT as TTC CAC AGG GCC GGG GGG C Real-time PCR (SEQ ID NO: 67)analysis of isoQC isoQCm MetI s ATG AGT CCC GGG AGC CGC Cloning of(SEQ ID NO: 68) murine isoQC cDNA isoQCm MetI as CTA GAG TCC CAG GTA CTCCloning of (SEQ ID NO: 69) murine isoQC cDNA isoQCm kurz sAGT TCC TGC CCC TGC TGC TG Cloning of (SEQ ID NO: 70) murine isoQC cDNAmQC RT s ATC AAG AGG CAC CAA CCA AC Real-time PCR (SEQ ID NO: 71)analysis of mQC mQC RT as CTG GAT AAT ATT TCC ATA G Real-time PCR(SEQ ID NO: 72) analysis of mQC mQC RT N-terminalACA GCT GGG AAT CTG AGT C Real-time PCR s analysis of mQC(SEQ ID NO: 73) mQC RT N-terminal GAG CAG AAT AGC TTC CGG GCGReal-time PCR as analysis of mQC (SEQ ID NO: 74) Iso-I55NsCTG CGG GTC CCA TTG AAC GGA AGC CTC CCC GAA Site-directed(SEQ ID NO: 75) mutagenesis hisoQC I55N Iso-I55NasTTC GGG GAG GCT TCC GTT CAA TGG GAC CCG CAG Site-directed(SEQ ID NO: 76) mutagenesis hisoQC I55N Iso-C351AsACG GTA CAC AAC TTG GCC CGC ATT CTC GCT GTG Site-directed(SEQ ID NO: 77) mutagenesis hisoQC C351A Iso-C351AasCAC AGC GAG AAT GCG GGC CAA GTT GTG TAC CGT Site-directed(SEQ ID NO: 78) mutagenesis hisoQC C351A hQC-1ATATATAAGCTTATGGCAGGCGGAAGACAC Insertion of (SEQ ID NO: 82)native hQC into pcDNA 3.1 hQC-2 ATATGCGGCCGCTTACAAATGAAGATATTCCInsertion of (SEQ ID NO: 83) native hQC into pcDNA 3.1 hisoQC pcDNA asATATATGCGGCCGCCTAGAGCCCCAGGTATTCAGC Amplification (SEQ ID NO: 84) hisoQCincluding the stop codon for insertion into pcDNA 3.1 EGFP-1ATATCTCGAGTCCATCGCCACCATGGTGAGC Amplification (SEQ ID NO: 85) EGFPEGFP-2 ATATCTCGAGTTACTTGTACA GCTCGTCCAT Amplification (SEQ ID NO: 86)EGFP hisoQC SS EGFP ATATGCGGCCGCATGTCGACGCTCCAAATGGTGTAGAACGCAmplification pcDNA as hisoQC (SEQ ID NO: 87) N-terminal sequencehQC C-FLAG ATATGCGGCCGCTTACTTGTCATCGTCATCCTTGTAATC AmplificationpcDNA as CAAATGAAGATATTCCAA hQC C-FLAG (SEQ ID NO: 88) hisoQC C-FLAGATATGCGGCCGCCTACTTGTCATCGTCATCCTTGTA Amplification h- pcDNA asATCGAGCCCCAGGTATTCAGC isoQC C-Flag (SEQ ID NO: 89) Hs_QPCT_1_SGQuantiTect Primer Assay (200), Qiagen, Hilden qPCR hQC Hs_QPCTL_1_SGQuantiTect Primer Assay (200), Qiagen, Hilden qPCR h-isoQC CCL2-FGCCTCCAGCATGAAAGTCTC qPCR CCL2 (SEQ ID NO: 90) CCL2-RCAGATCTCCTTGGCCACAAT (SEQ ID NO: 91) CCL7-F ATGAAAGCCTCTGCAGCACTqPCR CCL7 (SEQ ID NO: 92) CCL7-R TGGCTACTGGTGGTCCTTCT (SEQ ID NO: 93)CCL8-F TCACCTGCTGCTTTAACGTG qPCR CCL8 (SEQ ID NO: 94) CCL8-RATCCCTGACCCATCTCTCCT (SEQ ID NO: 95) CCL13-F ATCTCCTTGCAGAGGCTGAAqPCR CCL13 (SEQ ID NO: 96) CCL13-R AGAAGAGGAGGCCAGAGGAG (SEQ ID NO: 97)HIF1α-F CACAGAAATGGCCTTGTGAA qPCR HIF1α (SEQ ID NO: 98) HIF1α-RCCAAGCAGGTCATAGGTGGT (SEQ ID NO: 99) AIM1-F TCCTTTCATCCTGGAACCTGqPCR AIM1 (SEQ ID NO: 100) AIM1-R CGCCTCTTCTGTTTCACCTC (SEQ ID NO: 101)AIM2-F AAGCGCTGTTTGCCAGTTAT qPCR AIM2 (SEQ ID NO: 102) AIM2-RCACACGTGAGGCGCTATTTA (SEQ ID NO: 103) MAGEA1-F GTCAACAGATCCTCCCCAGAqPCR MAGEA1 (SEQ ID NO: 104) MAGEA1-R CAGCATTTCTGCCTTTGTGA(SEQ ID NO: 105) MAGEA2-F AGGTGGAGAGCCTGAGGAAT qPCR MAGEA2(SEQ ID NO: 106) MAGEA2-R CTCGGGTCCTACTTGTCAGC (SEQ ID NO: 107)MAGEA10-F AAGCGAGGTTCTCGTTCTGA qPCR (SEQ ID NO: 108) MAGEA10 MAGEA10-RTGACCTCTTGCTCTCCCTGT (SEQ ID NO: 109) MAGEB2-F CTTCAAGCTCTCCTGCTGCTqPCR MAGEB2 (SEQ ID NO: 110) MAGEB2-R CGACCCTGACTTCCTGGTTA(SEQ ID NO: 111) MART1-F GCTCATCGGCTGTTGGTATT qPCR MART1(SEQ ID NO: 112) MART1-R ATAAGCAGGTGGAGCATTGG (SEQ ID NO: 113) MCL1-FATGCTTCGGAAACTGGACAT qPCR MCL1 (SEQ ID NO: 114) MCL1-RATGGTTCGATGCAGCTTTCT (SEQ ID NO: 115) TYR-F TACGGCGTAATCCTGGAAACqPCR TYR (SEQ ID NO: 116) TYR-R ATTGTGCATGCTGCTTTGAG (SEQ ID NO: 117)TYRP1-F CCGAAACACAGTGGAAGGTT qPCR TYRP1 (SEQ ID NO: 118) TYRP1-RTCTGTGAAGGTGTGCAGGAG (SEQ ID NO: 119) TYRP2-F GGTTCCTTTCTTCCCTCCAGqPCR TYRP2 (SEQ ID NO: 120) TYRP2-R AACCAAAGCCACCAGTGTTC(SEQ ID NO: 121)

TABLE 5 Purification of GST-isoQC fusion protein following Expression inE. coli. Purification Step 1 2 3 4 Method Ni²⁺-IMAC GST-TAG AC GF IEX(EBA) (Desalting) (UNO S) Column type Chelating Glutathion Sephadex“continuous (Amersham Sepharose Sepharose G-25 Fine bed” matrixBiosciences AB, Fast Flow 4 Fast Flow BIO-Rad Sweden) Column size d =2.5 cm d = 1.6 cm d = 2.6 cm d = 1.2 cm l = 42 cm l = 10 cm l = 10 cm l= 5.3 cm CV = 206 cm³ CV = 20 cm³ CV = 53 cm³ CV = 6 cm³ EquilibrationBuffer PBS PBS 25 mM Mes 25 mM Mes pH 7.3 7.3 6.0 6.0 Volume 10 CV 10 CV10 CV 10 CV Intermediate (Wash) Buffer PBS PBS — 25 mM Mes 0.5 mMHistidin pH 7.3 7.3 6.0 Volume 10 CV 10 CV 10 CV Elution Buffer PBS 50mM Tris 25 mM Mes 25 mM Mes 100 mM Histidin 10 mM Gradient Glutathionelution NaCl (reduced) pH 7.3 8.0 6.0 6.0 Volume 1.5 CV  (reverse flow) 1 CV CV The purified fusion protein was used for determination of QCactivity.

TABLE 6 K_(i)-values for competitive inhibition of human QC and humanisoQC by imidazole derivatives. Ki (μM) Ki (μM) Ki (μM) InhibitorhisoQCdt YSShisoQC hQC Imidazole 220 ± 1  235 ± 13  103 ± 2 Benzimidazole 200 ± 8  250 ± 5  138 ± 4  1-Benzylimidazole 7.3 ± 0.5 6.2± 0.2 7.1 ± 0.1 1-Methylimidazole 80 ± 5  82 ± 3  39.7 ± 0.2  PBD1501-(3,4- 0.48 ± 0.03 0.519 ± 0.001 0.0584 ± 0.0002 Dimethoxy- phenyl)-3-(3-imidazole-1-yl- propyl)-thiourea Human isoQC was expressed in E. coliBL21 (hisoQCdt) or P. pastoris (YSShisoQC).

Example 6 Expression and Purification of Human isoQC in P. Pastoris HostStrains and Media

Escherichia coli strain DH5α was used for propagation of plasmids and P.pastoris strain X-33 was used for the expression of human isoQC inyeast. E. coli and P. pastoris strains were grown, transformed andanalyzed according to the manufacturer's instructions (Qiagen (DH5α),invitrogen (X-33)). The media required for E. coli, i.e. Luria-Bertani(LB) medium, was prepared according to the manufacturer'srecommendations. The media required for Pichia pastoris, i.e. BMMY,BMGY, YPD, YPDS and the concentration of the antibiotics, i.e. Zeocin,were prepared as described in the Pichia manual (invitrogen, catalog.No. K1740-01). The manual also includes all relevant descriptions forthe handling of yeast.

Molecular Cloning of Plasmid Vectors Encoding the Human QC

All cloning procedures were done applying standard molecular biologytechniques. For expression in Pichia pastoris X-33, the pPiCZαA(invitrogen) was used. The cDNA of the mature human isoQC starting withcodon 30 (counting from methionine II) was fused in frame with theplasmid encoded α-factor, directing the protein into the secretorypathway. After amplification utilizing the primers hisoQC HIS C-TermpPICZAA-1 (SEQ ID NO: 62) or hisoQC HIS N-Term pPICZAA-1 (SEQ ID NO: 63)as sense-Primers and hisoQC HIS N-Term pPICZAA-2 (SEQ ID NO: 64) andhisoQC HIS C-Term pPICZAA-2 (SEQ ID NO: 66) (Table 4) as antisensePrimers, the fragment was inserted into the expression vector employingthe restriction sites of NotI and EcoR I. Depending on the construct,Mutations were introduced in codons 55 (Ile) and 351 (Cys). Themutagenesis was performed according to standard PCR techniques followedby digestion of the parent DNA using DpnI (quik-change II site-directedmutagenesis kit, Stratagene, Catalog No. 200524). The generatedconstructs are illustrated schematically in FIG. 17.

Transformation of P. pastoris and Mini-Scale Expression

1-2 μg of plasmid DNA were applied for transformation of competent P.pastoris cells by electroporation according to the manufacturer'sinstructions (BioRad). Selection was done on plates containing 100 μg/mlZeocin. In order to test the recombinant yeast clones upon is QCexpression, recombinants were grown for 24 h in 10 ml conical tubescontaining 2 ml BMGY. Afterwards, the yeast was centrifuged andresuspended in 2 ml BMMY containing 0.5% methanol. This concentrationwas maintained by addition of methanol every 24 h for about 72 h.Subsequently, QC activity in the supernatant was determined. Clones thatdisplayed the highest activity were chosen for further experiments andfermentation. Depending on the expressed construct, the isoQC-activityin the medium differed (FIG. 18).

Expression and Purification of hisoQC in P. pastoris

For large scale-Expression of isoQC in Pichia pasoris, the conditionwere kept as described in the mini-scale expression, however, the totalvolume was 8 L. The expression was performed in shake-flasks. Afterexpression, cells were separated from the medium by centrigugation(1500×g, 20 min), and the pellet discarded. The pH-value of thesupernatant was adjusted to neutrality, centrifuged again and appliedfor the first purification step. The isoQC protein was purifiedutilizing a 3-step protocol (Table 7). The purfication is illustrated bySDS-PAGE analysis in FIG. 19.

TABLE 7 Purification of hisoQC (YSShisoQCN55IC351A C- His) followingExpression in P. pastoris. Purification Step 1 2 3 Method Ni²⁺-IMAC HICGF (Desalting) Column type Chelating Butyl Sepharose Sephadex G-25(Amersham Sepharose 4Fast Flow Fine Biosciences Fast Flow AB, Sweden)Column size d = 2.5 cm d = 1.6 cm d = 2.6 cm l = 42 cm l = 15.5 cm l =10 cm CV = 206 cm³ CV = 23 cm³ CV = 53 cm³ Equilibration Buffer 50 mMNaH₂PO₄ 30 mM NaH₂PO₄ 50 mM Bis-Tris 1M (NH₄)₂SO₄ 100 mM NaCl pH 7.0 7.06.8 Volume 10 CV 10 CV  10 CV Intermediate (Wash) Buffer 50 mM NaH₂PO₄30 mM NaH₂PO₄ — 0.5 mM Histidin 1M (NH₄)₂SO₄ pH 7.0 7.0 Volume 10 CV 6CV Elution Buffer 50 mM NaH₂PO₄ 30 mM NaH₂PO₄ 50 mM Bis-Tris 100 mMHistidin 100 mM NaCl pH 7.0 7.0 6.8 Volume 1.5 CV  5 CV  1 CV Thepurified fusion protein was used for determination of QC activity andpH-dependence.

Results

Human isoQC was expressed in the methylotrophic yeast P. pastorissuccessfully. Several different constructs were generated, in order toselect the best expression conditions in yeast (FIG. 17). As illustratedin FIG. 18, the QC activity that is expressed and present in the mediumof the expressing cells, varies depending on the expressed construct.Introduction of a glycosylation site resulted in proper secretion, ascan be observed from constructs YSShisoQCN55IC351A C-His andYSShisoQCN55I C-His. Due to the highest activity in the medium,construct YSShisoQCN55IC351A C-His was expressed in large-scale andpurified. The purification was carried out as described in Table 7, theyield of purification was 59%. The apparent homogeneous protein wasglycosylated, as evidenced by a shift in migration to lower molecularmass (FIG. 19). Glycosylation did not influence the catalytic activityof the enzyme.

Example 7 The pH-Dependence of hisoQC

The fluorometric assay using H-Gln-βNA (described in example 5) wasapplied to investigate the pH-dependence of the catalytic specificity.The reactions were carried out at substarte concentrations of 7 μM, i.e.at [S]<<K_(M). Therefore, the observed specificity constants could bedirectly deduced from the initial velocity of the progress curves ofsubstrate conversion. In these studies the reaction buffer consisted of0.075 M acetic acid, 0.075 M Mes and 0.15 M Tris, adjusted to thedesired pH using HCl or NaOH. The buffer assures a constant ionicstrength over a very broad pH-range. Evaluation of the acquired enzymekinetic data was performed using the following equation:

k _(cat) /K _(M)(pH)=k _(cat) /K _(M)(limit)*1/(1+[H ⁺ ]/K _(HS) +K_(E1) /[H ⁺ ]+K _(E1) /[H ⁺ ]*K _(E2) /[H ⁺]),

in which k_(cat)/K_(M) (pH) denotes the pH-dependent (observed) kineticparameter. k_(cat)/K_(M) (limit) denotes the pH-independent (“limiting”)value. K_(HS), K_(E1) and K_(E2) denote the dissociation constants of andissociating group in the acidic pH-range, and two dissociating groupsof the enzyme, respectively. Evaluation of all kinetic data wasperformed using GraFit software (version 5.0.4. for windows, ERITHACUSSOFTWARE Ltd., Horley, UK).

Results

The hisoQC displays a pH-optimum of specificity at pH 7-8. Thus, thepH-optimum of catalysis is very similar to human QC. Fitting of the dataaccording to a model which is based on three dissociating groupsresulted in a well interpretation of the pH-dependence of hisoQC and hQC(FIG. 22). Thus, the catalysis of both enzymatic reactions is influencedby similar dissociating groups, suggesting a similar catalytic mechanismin general.

The determined pKa-values are displayed in Table 8. It is obvious, thatonly one pKa differs between hisoQC and hQC significantly. In hQC, thepKa corresponds to the pKa of the dissociation constant of thesubstrate. Possibly, the subtle difference between hQC and hisoQC iscaused by structural changes occurring in isoQC catalysis (induced fit),influencing the pH-dependence.

Example 8 Investigation of Glutamyl Cyclase Activity

It has been described for human QC, that the enzyme catalyses thecyclization of N-terminal glutamic acid into pyroglutamic acid.Therefore, QC is involved inteo the generation of pGlu-modified amyloidpeptides.

In order to investigate the cyclization of glutamic acid, human QC andhuman isoQC were purified and the formation of pGlu-modified amyloidβ(3-11) [pGlu-Aβ(3-11)] from Aβ(3-11) was monitored. Reactions consistedof 20 μl substrate (Aβ(3-11), 2.5 mM stock solution in 50 mM Mes buffer,pH 6.5) and 80 μl enzyme (0.62 mg/ml hQC stock solution; 0.61 mg/mlhisoQC stock solution in 50 mM Mes pH 6.5). Samples (15 μl) were removedafter 0 h, 6 h, 24 h, 48 h and 72 h and boiled for 5 min in order toterminate the reaction. The analysis of substrate conversion wasmonitored by Maldi-T of mass spectrometry. Substrate and product differin their molecular mass by 18 Da, the mass of water, which is releasedduring cyclization.

As shown in FIG. 23, human QC and human isoQC (YSShisoQCI55NC351A C-His)catalyze the conversion of Aβ(3-11) into pGlu-Aβ(3-11). However, basedon equal protein concentrations in both samples, one can conclude thatthe conversion of N-terminal glutamic acid by hisoQC is much slowercompared with hQC. Thus, the lower specificity constants for conversionof glutaminyl substrates is also observed with glutamyl substrates. Nocyclization was observed under these conditions with inactivated enzyme(Schilling, S. et al., 2004 FEBS Lett. 563, 191-196).

Example 9 Tissue Specificity of Murine isoQC

The tissue distribution of murine QC and murine isoQC was investigatedusing quantitative real time PCR techniques. Prior to analysis of cDNAfrom several different organs and tissues, the murine isoQC open readingframe was isolated applying specific primers (isoQCm MetI s (SEQ ID NO:68), isoQCm MetI as (SEQ ID NO: 69) (table 4), which were deduced fromthe chromosomal coding region of murine isoQC.

The open reading frame was cloned into vector pPCR-Script CAM SK (+)(PCR-Script CAM Cloning Kit, Stratagene) and used as a positive controlin the real-time PCR determinations and for preparation of a standardcurve under assay conditions.

The characterization of the tissue specificity of misoQC expression wasachieved applying cDNA from 3-6 month old mice. Total RNA was isolatedfrom 30 mg tissue, using the RNA-isolation kit II (Macherey and Nagel).The RNA concentration and purity was assessed by gelelectrophoresis(agarose gel) and spectrophotometry. For synthesis of cDNA, 1 μg of RNAwas used. The reaction was done applying the reverse TranscriptaseSuperscript II RT (Invitrogen) according to the recommendations of thesupplier, the cDNA was stored at −80° C.

The quantitative analysis of the transcript concentration in differenttissues was analysed using the “Light Cycler” (Corbett research),applying the “QuantiTect SYBR Green PCR” (Qiagen). The DNA standard(cloned cDNA isoQC mouse) was used for quantification. The copy numberwas calculated according to the following equation: (X^(g)/_(μl)DNA)/(Plasmid length in bp*660)*6.022*1023=Y^(Molecules)/_(μl). The DNAstandard contained 4 concentrations in the range of10⁷-10^(1 Molecules)/_(μl), and an limiting concentration (10⁰). Thereaction protocoll is displayed in Table 8. The results are displayed inFIG. 24.

For amplification of murine QC, the same protocol was used, applying theprimers mQC RT N-terminal s (SEQ ID NO: 73) and mQC RT N-terminal as(SEQ ID NO: 74).

TABLE 8 Reaction protocol of the quantitative real-time-PCR using theRoto-Gene RG 3000 (Corbett Research) PCR-Cycles step T in ° C. t in sec.0 Denaturation 95 900 1 Denaturation 95 15 2 Primer Annealing 55 20 3Elongation 72 20 Cycles 45

Results

As shown in FIG. 24, murine QC and murine isoQC are expressed in allorgans tested. In contrast to murine QC, the variances in expression ofmurine isoQC between different organs are smaller, indicating a lowerstringency of regulation of transcription. The data for expression ofmQC correspond to previous analyses of bovine QC, which was analyzedusing Northern-Blot (Pohl, T. et al. 1991 Proc Natl Acad Sci USA 88,10059-10063). Highest expression of QC was observed in Thalamus,Hippocampus and Cortex. Thus, QC-expression is primarily detected inneuronal tissue. Little QC-expression is detected in peripheral organsas spleen and kidney. Also misoQC is expressed in neuronal tissue, butat lower levels compared with mQC. In contrast, expression levels inperipheral organs is very similar between isoQC and QC.

Concluding, based on the results of transcript concentration, thecombined activity (isoQC and QC) should be highest in brain. Thus,highest QC-protein levels are present in organs that are afflicted byamyloidoses like Alzheimers Disease, familial british dementia andfamilial danish dementia.

Example 10 Inhibition of Human isoQC by Heterocyclic Chelators Results

The time-dependent inhibition of QCs from different sources usingheterocyclic chelators, such as 1,10-phenanthroline and dipicolinic acidhas been investigated previously (6, 9). In analogy, h-isoQC is alsotime-dependently inactivated by the heterocyclic chelators1,10-phenanthroline (FIG. 25) and dipicolinic acid (not shown), clearlypointing to a metal-dependent activity. Furthermore, EDTA also inhibitedh-isoQC (FIG. 25). This is in sharp contrast to QCs, since neither humanQC, porcine QC nor murine QC has shown discernible inhibition by EDTA.However, inhibition of hisoQC by EDTA even stronger suggests ametal-dependent catalysis.

Example 11 Subcellular Localization of hisoQC Investigated Using CellFractionation Cell Fractionation

The day following transfection, expressing HEK293 cells were washed withD-PBS and collected by centrifugation at 500×g for 5 min at 4° C.Subsequently, D-PBS was discarded and the cells were resuspended in 1 mlof disruption buffer (50 mM Tris, 50 mM KCl, 5 mM EDTA, 2 mM MgCl₂, pH7.6 adjusted with HCl) and cracked by 30 crushes in a Potter cellhomogenisator. The suspension was centrifuged at 700×g for min at 4° C.The obtained pellet was resuspended in 300 μl disruption buffer anddesignated as debris fraction (D). The resulting supernatant was furthercentrifuged at 20.000×g for 30 min at 4° C. The pellet illustrated theheavy membrane fraction (HM) and was resuspended in 200 μl disruptionbuffer. The resulting supernatant was centrifuged at 100.000×g for 1 hat 4° C. using an ultracentrifuge (Beckmann). The obtained pellet wasresuspended in 200 μl disruption buffer and was termed as light membranefraction (LM). The supernatant was designated as soluble fraction (S).Debris, heavy membrane and light membrane fractions were sonicated for10 sec and. the protein content of all fractions was determined usingthe method of Bradford. Subsequently, fractions were analyzed for QCactivity and stained for marker proteins using Western Blot.

Results

For further corroboration, biochemical analysis of QC activitydistribution, derived from hisoQC and hQC expression were performed. Thenative hisoQC beginning with methionine I and II and hQC were expressedin HEK293 cells, respectively. After cell fractionation the QC activityin the each fraction was determined using the fluorescence assayapplying H-Gln-βNA as substrate. In cells, transfected with the emptyvector (pcDNA), specific QC activity is hardly measurable. Whenexpressing native hisoQC (MetI) and hisoQC (MetII), QC activity wasreadily detectable with the highest specific activity in the heavymembrane fraction (MetI: 40±2 μmole/min/g; MetII: 36±1.5 μmole/min/g)and the medium (MetI: 30±2 μmole/min/g; MetII: 54±3 μmole/min/g). Incontrast, hQC shows the highest specific QC activity within the medium(1339±76 μmole/min/g) followed by the heavy membrane fraction (251±21μmole/min/g) (FIG. 26A).

In addition the absolute activities were calculated, illustrating thatthe expression of hisoQC (MetI) and hisoQC (MetII) led mainly to anincrease in the intracellular QC activity, namely within the debris(MetI: 1032±9 nM/min; MetII: 1110±10 nM/min) and heavy membrane fraction(MetI: 374±20 nM/min; MetII: 281±12 nM/min). Only little QC activity wasfound within the medium (MetI: 27±2 nM/min; MetII: 53±3 nM/min). Incontrast, QC activity deduced by hQC expression shows high activitywithin the medium (1138±65 nM/min) and within intracellular compartments(debris: 1089±14 nM/min; heavy membrane fraction: 583±38 nM/min)supporting an Golgi localization of hisoQC as shown by histochemicalanalysis (FIG. 26B).

The data obtained by the expression of the native enzymes was furthersupported by expression of hisoQC (MetI and MetII) and hQC possessing aC-terminal FLAG-tag (FIG. 26C). Western Blot analysis of the resultingFLAG-tagged proteins in comparison to marker proteins of the Golgicomplex and mitochondria revealed a mainly intracellular localization ofhisoQC(MetI) and hisoQC (MetII) within the debris and heavy membranefraction, whereas hQC is enriched within the medium but also foundwithin the debris and heavy membrane fraction. Visualization of markerproteins of the Golgi complex (ST1GAL3) and mitochondria revealed thepresence of these compartments within the debris and heavy membranefraction. In addition the 65 kDa mitochondrial protein was also found toa smaller portion within the soluble fraction.

Example 12 Analysis on the Golgi Retention Signal of hisoQC

In order to clarify, whether the predicted N-terminal transmembranehelix is responsible for the retention of hisoQC within the Golgicomplex, the signal peptides starting at MetI and MetII, including thetransmembrane helix, were cloned in frame with EGFP. The resultingvectors hisoQC (MetI) SS EGFP and hisoQC (MetII) SS EGFP were expressedin LN405 cells and examined in analogy to the full-length hisoQC EGFPfusion proteins using confocal laserscanning microscopy. The expressionof hisoQC (MetI) SS EGFP led to the same Golgi complex localizationobserved for the full-length hisoQC (MetI) EGFP fusion protein. Again, atransport of hisoQC (MetI) SS EGFP to the mitochondria was not observed(FIG. 27A). In addition, the expression of the N-terminal truncatedpeptide hisoQC (MetII) SS EGFP also led to a enrichment of the proteinwithin the Golgi complex. In analogy to hisoQC (MetI) SS EGFP, nomitochondrial EGFP fluorescence could be recorded (FIG. 27B).Consequently, the N-terminal sequence of hisoQC leads to theco-translational translocation of the protein to the ER membrane and tothe retention within the Golgi complex. Furthermore, due to theexpression of hisoQC (MetII) SS EGFP, the Golgi retention signal wasgrossly mapped to reside between methionine 19 and serine 53 (countingof amino acids beginning at MetI).

Additional topology analysis revealed the possibility for a functionalhomology of the hisoQC N-terminus to glycosyltransferases.Glycosyltransferases are type II transmembrane proteins, possessing ashort cytoplasmatic sequence, followed by the transmembrane helix and alarge luminal catalytic domain. Clearly, this is essentially the samedomain structure as found for misoQC and hisoQC (FIG. 28). For a numberof glycosyltransferases, the Golgi retention signal was identified toreside within the transmembrane domain. Furthermore, for some of theseenzymes truncation of the cytoplasmatic sequence was found to have noinfluence on the activity or the localization of the protein. Insummary, evidence was provided, that hisoQC is a type II transmembraneprotein showing a retention within the Golgi complex similar toglycosyltransferases.

Example 12 Detection of QPCTL mRNA in Different Human Carcinoma CellLines and Tissues

qPCR Analysis

Analysis of human QPCTL expression in human carcinoma cell lines wereperformed using the quantitative real time PCR (qPCR) technique,essentially as described in example 9. For determining QPCTL mRNA,primers of the QuantiTect® primer assay were applied covering anexon/exon region for exclusion of co-amplification of genomic DNA. QPCRwas performed following the manufacturers recommendations. The reactionmixture is depicted in Table 9 and the PCR program is illustrated inTable 8.

TABLE 9 Composition of the qPCR mixture component Volume in μl 2xQuantiTect SYBR Green PCR Master Mix 7.5 (2.5 mM MgCl₂) 10x QuantiTectPrimer Assay 1.5 cDNA (≦100 ng/Reaktion) 1 Aqua bidest. 5

The quantitative analysis of the transcript concentration in differenttissues was analysed using the “Light Cycler” (Corbett research),applying the “QuantiTect SYBR Green PCR” (Qiagen). The DNA standard(cloned cDNA isoQC human) was used for quantification. The copy numberwas calculated according to the following equation: (X^(g)/_(μl)DNA)/(Plasmid length in bp*660)*6.022*1023=Y^(Molecules)/_(μl). The DNAstandard contained 4 concentrations in the range of10⁷-10^(1 Molecules)/_(μl), and an limiting concentration (10⁰).

The results of qPCR were evaluated using the rotor-gene operatingsoftware (Corbett research).

Results Expression of QPCTL in Different Carcinoma Cell Lines

Among the tested cancer cell lines, human melanoma cells show thehighest expression of QPCTL transcripts (approx. 7000 copies/50 ngtotal-RNA), whereas the human soft tissue sarcoma cell lines show thelowest expression of QPCTL (365 copies/50 ng total-RNA). Pancreascarcinoma shows 2100 copies, thyroid carcinoma 3500 copies and gastriccarcinoma possesses 4100 copies in the median (FIG. 29).

Expression of QPCTL in Different Melanoma Cell Lines

Recently it has been shown, that melanoma cells possess comparable highQPCT expression (Gillis, J. S., J. Transl. Med. 4 (2006), 4:27).Therefore, QPCTL expression in different melanoma cell lines wasanalyzed. As depicted in FIG. 30, QPCTL expression was detected in allmelanoma cell lines, tested. The variation among the cell lines variedfrom 2025 copies/50 ng total-RNA in line Mel_ZL_(—)11 to 18043 copies/50ng total-RNA in line Mel_ZL12.

TABLE 10 Correlation of QPCT and QPCTL to tumor-associates antigens(taa) and correlation of taa among each other correlation significancecorrelation significance QPCT - MAGEB2 0.0436 AIM1 - MCL1 0.0163 QPCT -MART1 0.0020 MAGEA1 - MAGEA2 0.00002 QPCT - TYR 0.0023 MAGEA1 - MAGEB20.0058 QPCT - MAGEA1 0.0591 TYRP2 - MART1 0.0042 QPCTL - MART1 0.0008TYR - MART1 0.0335 TYR - TYRP2 0.0408 AIM1 - AIM2 0.0082 TYR - MCL-10.0151

Furthermore, QPCT and QPCTL expression was correlated to the expressionof tumor-associated antigens (taa). The melanoma-specifictumor-associated antigens were selected by data base mining andpublished results. Among others, AIM1 and AIM2 (absent in melanoma),MAGEA1, -A2, -A10 and MAGEB2 (melanoma antigen family A and B), MART1(melanoma antigen recognized by T-cells), TYR (tyrosinase), TYRP1 andTYRP2 (tyrosinase related protein) and MCL-1 (myeloid cell leukemia) aretumor-associated antigens in melanoma. Data were compared using SPSSstatistic software. Correlation between QPCT and MAGEB2 was significant(p=0.0436). Furthermore, correlation between QPCT and MART1 (p=0.002),QPCTL and MART1 (p=0.008) and QPCT and TYR (p=0.0023) was alsostatistically highly significant. The correlations show a directdependence, which implies: the higher QPCT/QPCTL expression, the higherthe expression of tumor-associated antigens. The only exception is thecorrelation between TYR and MCL1, which shows an indirect dependence.

Expression of QPCT and QPCTL in Different Tumor Tissues

The expression of QPCT and QPCTL was evaluated in tumor tissues of softtissue sarcoma, gastric carcinoma and thyroid carcinoma. Highestexpression of QPCT has been found in thyroid carcinoma followed bygastric carcinoma and soft tissue carcinoma (Table 11). The same orderwas observed for QPCTL expression, however, the copy number of QPCTLtranscripts was always lower, than observed for QPCT transcripts asrevealed by Student's t-test (p_(soft tissues carcinoma)=0.001;P_(gastric carcinoma)=4.8E-7; p_(thyroid carcinoma)=0.04) (Table 11;FIG. 31).

TABLE 11 Comparison of QPCT and QPCTL expression in different tumortissues soft tissue sarcoma gastric carcinoma thyroid carcinoma (119samples) (47 samples) (29 samples) QPCT 1293 2985 8303 QPCTL 170 4692540

Further investigations on the expression level of QPCT and QPCTLrevealed a two-sided significant correlation by Pearson in soft tissuesarcoma (p=2E-31) and gastric carcinoma (p=0.015). No correlation hasbeen observed for QPCT and QPCTL expression level in thyroid carcinoma(p=0.46).

Expression of QPCTL Dependent on the Stage of Differentiation in GastricCarcinoma

For gastric carcinomas, QPCTL expression in samples representingdifferent stages of tumor differentiation were investigated. As controlserved tumor-surrounding normal tissue. The comparison of normal withtumor tissue revealed a significantly higher QPCTL expression (p=0.04)in tumor tissues. Undifferentiated gastric carcinomas show lower QPCTLexpression, than normal tissue. Poorly and well to moderatedifferentiated gastric carcinomas show no differences in the mediancompared to normal tissue (FIG. 32).

Expression of QPCT and QPCTL in Different Stages of Thyroid Carcinoma

Different stages of thyroid carcinoma were inverstigated concerning QPCTand QPCTL expression. The stages were classified according tonomenclature of the world health organisation (WHO) as follicularthyroid carcinoma (FTC), papillary thyroid carcinoma (PTC) andundifferentiated thyroid carcinoma (UTC). Samples from patientspossessing goiter served as control.

The QPCT mRNA level (median) in differentiated thyroid carcinomas FTC(6700 copies/50 ng total-RNA) and PTC (16000 copies/50 ng total-RNA)were higher than in non-tumor tissue (goiter: 2100 copies/50 ngtotal-RNA). UTC possesses 5400 copies/50 ng total-RNA and is 2.5 timeshigher than observed in goiter. The mRNA copy number of QPCT is in allthyroid tumors significantly higher than in goiter (p=0.04, Student'st-test) (FIG. 33).

The QPCTL mRNA level in thyroid carcinoma is homogeneous. The samplesfrom FTC (2600 copies/50 ng total-RNA) and UTC (2500 copies/50 ngtotal-RNA) are similar to goiter (2500 copies/50 ng total-RNA). Theexpression of QPCTL in PTC is slightly decreased to 1900 copies/50 ngtotal-RNA (FIG. 34).

In conclusion, QPCT and QPCTL are equally expressed in goiter. However,in tumor tissues the expression of QPCT increases, whereas theexpression of QPCTL remains stable.

Example 13 Investigations on the QPCT and QPCTL Expression in Human CellLines after Incubation with Different Stimuli Cell Lines and Media

The stimulation experiments were performed using the human embryonalkidney cell line HEK293, human acute monocytic leukemia cell line THP-1and the follicular thyroid carcinoma cell line FTC-133. Cells were grownin appropriate culture media (DMEM, 10% FBS for HEK293, RPMI1640, 10%FBS for THP-1 and DMEM/F12, 10% FBS for FTC-133) in a humidifiedatmosphere at 37° C. and 5% CO₂.

Stimulation Using Bioactive Peptides, Chemicals or LPS

HEK293 and FTC-133 cells were cultivated as adherent cultures and THP-1cells were grown in suspension. For stimulation assay 2×10⁵ cells ofFTC-133 and HEK293 cells were transferred to 24 well plates. In case ofHEK293, plates were coated with collagen I for ensuring properadherence. In addition, 2×10⁶ cells of THP-1 were grown in 24 wellsuspension plates. All stimulation experiments were applied underserum-free conditions. FTC-133 was grown over night. Afterwards, cellswere adapted to serum-free media for another 24 h and the stimulationwas started by replacing the conditioned media by fresh serum-freemedia. HEK293 cells were grown over night and afterwards the stimulationusing respective agents was started without an adaption to serum-freeconditions due to morphological changes in case of cultivation of HEK293under serum-free conditions for more than 24 h. THP-1 cells were platedin serum-free media together with respective agent. The applied stimuliand final concentrations are listed in Table 12.

TABLE 12 Stimuli for investigations on the regulation of hQC and hisoQCin human cell lines Name Final concentration butyric acid (BA) 2 mMhepatocyte growth factor (HGF) 10 ng/ml lipopolysaccharide (LPS) 1, 10μg/ml transforming growth factor β (TGFβ) 10, 100 ng/ml tumor necrosisfactor α (TNFα) 10, 100 ng/ml

Cells were incubated with the respective stimulus for 24 h. Afterwards,total-RNA from the cells was isolated using the Nucleo-Spin® RNA II Kit(Macherey-Nagel) and stored until qPCR assay.

Stimulation Using Hypoxia

THP-1, HEK293 and FTC-133 cells were plated into two 25 cm² tissueculture flasks, respectively. Thereby, one flask of each cell lineserved as negative control, cultivated under normal growth conditionsfor 24 h. The other flasks were placed in a anaerobic bag together withan anaerobic reagent (Anaerocult® P, Merck) and an indicator. The bagwas sealed to ensure air tight conditions. Cells were also grown for 24h and subsequently, total-RNA was isolated using the Nucleo-Spin® RNA IIKit (Macherey-Nagel) and stored until qPCR assay.

Results Basal Expression of QPCT and QPCTL in HEK293, FTC-133 and THP-1

The basal expression in the used cell lines HEK293, FTC-133 and THP-1was evaluated in preparation for the following stimulation experiments.The copy number of QPCT and QPCTL transcripts is summarized in Table 13.

TABLE 13 Basal expression of QPCT and QPCTL in different cell linesAbsolute mRNA copy numbers per 50 ng total RNA cell line QPCT QPCTLHEK-293 (8 samples)  37196 ± 18928 3206 ± 855 FTC-133 (8 samples) 24790± 7605 10262 ± 1899 THP-1 (8 samples) 3588 ± 853  6725 ± 1763

Influence of Selected Stimuli on Expression of QPCT and QPCTL

Regulational binding sites of the promotors of QPCT and QPCTL and signaltransduction pathways leading to their regulation are not described sofar. Therefore, stimulation experiments using different cell lines andstimuli were conducted. QPCT mRNA levels in HEK293 cells were increasedby stimulation using TNF-α, HGF and butyric acid. In addition theregulation of CCL2 as QPCT/QPCTL substrate has been inverstigated. TNF-αand butyric acid increased the amount of CCL2 transcripts in HEK293. HGFhad no influence in CCL2 expression. In contrast QPCTL was not regulatedby TNF-α, HGF and butyric acid (FIG. 35).

In addition FTC-133 was stimulated using LPS and TGF-β and theregulation of QPCT, QPCTL and CCL2 was monitored. In FTC-133, LPS andTGF-β stimulated the expression of QPCT mRNA, but failed to induce QPCTLand CCL2 expression (FIG. 36).

This experiments were further coroborated by stimulation of THP-1 cellsusing LPS (1 μg/ml), LPS (10 μg/ml), TGF-β and TNF-α. As observed forFTC-133 and HEK293, QPCT expression could be induced using differentstimuli. In addition CCL2 expression was induced using LPS and TNF-α.Again, no induction or repression of QPCTL mRNA could be observed (FIG.37).

In conclusion, the experiments revealed, that QPCT can be regulated by aset of stimuli in different cell lines (LPS, TNF-α, HGF, butyric acidand others). In contrast, QPCTL could neither stimulated nor repressedby the tested stimuli suggesting a house-keeping function of QPCTL.

Influence of Selected Stimuli on Expression of QPCT its Substrates

Since QPCT expression was induced by a number of stimuli, the questionwas raised, whether QPCT induction takes place in combination with aninduction of the QPCT substrates CCL2, CCL7, CCL8 and CCL13. Therefore,the stimulation using LPS (1 μg/ml), LPS (10 μg/ml), TGF-β (100 ng/ml)and TNF-α (100 ng/ml), respectively, was performed using THP-1monocytes. THP-1 expresses all chemokines at a basal level, importantfor comparison of stimulated cells with the negative control.

LPS and TNF-α led to the reliable induction of all tested chemokines andQPCT in THP-1 cells. TGF-β was less effective as stimulus and inducedthe expression of QPCT, CCL2, CCL7 and CCL8 maximum 2 fold. CCL13 wasrepressed by TGF-β stimulation (FIG. 38).

Stimulation of QPCT and QPCTL Expression by Hypoxia

QPCTL expression could not be regulated by chemical agents, bioactivepepitides or LPS. Therefore, we tested, whether QPCTL expression isregulated by hypoxia. As summarized in FIG. 39. Hypoxia selectivelyinduced the expression of QPCTL but not of QPCT. In comparison, hypoxiainduced factor 1a (HIF1a) was repressed by 15% (FIG. 39A) and 45% (FIG.39C). The data suggest a connection of QPCTL to hypoxia.

Synthesis of the Inhibitors

Analytical Conditions

ESI-Mass spectra were obtained with a SCIEX API 365 spectrometer (PerkinElmer). The ¹H-NMR (500 MHz) data was recorded on a BRUKER AC 500, usingDMSO-D₆ as solvent. Chemical shifts are expressed as parts per milliondownfield from tetramethylsilane. Splitting patterns have beendesignated as follows: s (singulet), d (doublet), dd (doublet ofdoublet), t (triplet), m (multiplet), and br (broad signal).

Detailed Synthesis Description Examples 1-12 and 14-53

1H-imidazole-1-propanamine was reacted with the correspondingisothiocyanate in ethanol under reflux for 8 h. After that the solventwas removed and the remaining oil was dissolved in methylene chloride.The organic layer was washed twice with a saturated solution of NaHCO₃followed by NaHSO₄ and brine, dried then evaporated. The remaining solidwas re-crystallized from ethyl acetate, yielding the example thiourea inyields of 80-98%.

Example 131-(3-(1H-imidazol-1-yl)propyl)-3-(3,4-dimethoxyphenyl)thiourea

4.0 mmol of 3,4-dimethoxyphenyl isothiocyanate and 4.0 mmol of3-(1H-imidazol-1-yl)alkyl-1-amine were dissolved in 10 mL of absoluteethanol. After stirring for 2 h under reflux, the solvent was evaporatedand the resulting solid was recrystallized from ethanol.

Yield: 0.66 g (51.3%); mp: 160.0-161.0° C.

¹H NMR δ 1.8-2.0 (m, 2H), 3.4-3.5 (m, 2H), 3.75 (s, 6H), 3.9-4.0 (m,2H), 6.7-6.8 (m, 1H), 6.9 (br m, 2H), 6.95 (s, 1H), 7.15 (s, 1H), 7.55(br s, 1H), 7.6 (s, 1H), 9.3 (s, 1H); MS m/z 321.2 (M+H), 253.3(M-C₃H₃N₂.)

Examples 96-102

1H-imidazole-1-propanamine was reacted with the corresponding isocyanatein ethanol under reflux for 8 h. After that the solvent was removed andthe remaining oil was dissolved in methylene chloride. The organic layerwas washed twice with a saturated solution of NaHCO₃ followed by NaHSO₄and brine, dried then evaporated. The remaining solid wasre-crystallized from ethyl acetate, yielding the example urea in yieldsof 85-90%.

Examples 136, 137

The 1H-imidazole-1-alkylamines were prepared according to the literaturefrom-brom-alkyl-phtalimides and imidazolium salt and subsequenthydrazinolysis. The resulting products were transformed into thethioureas according to example 1-53 giving a 88% (example 136) and 95%(example 137) yield.

Examples 54-95

All examples were made from the corresponding thioureas by reacting withWater-soluble-carbodiimide (WSCD) and 1H-imidazole-1-propanamine in drydimethyl form-amide for 2 h at r.t. giving the trisubstituted guanidineswith yields from 40-87%.

Examples 103-105

Imidazole was reacted with the corresponding brommethylphenylcyanide inDMF, utilizing 1 equivalent of NaH for 3 h under rt., giving the1H-imidazole-1-methylphenylcyanides. The solvent was removed and theresulting oil was re-dissolved in dioxane. The cyanides were convertedin the corresponding amines using 1 equivalent of LiAlH₄. After adding asaturated solution of KHSO₄, dioxane was evaporated and the aqueouslayer was extracted by means of CHCl₃. The organic layer wasconcentrated in vacuo and the amine was converted in the correspondingthioureas according to example 1-53 giving a 78% (example 103) and 65%(example 104) and 81% (example 105) yield.

Examples 106-109

Starting from the correspondingmethansulfonate-2-methylpropyl-phthalimides the amines were synthesizedas described for the amines in example 136-137. The resulting productswere transformed into the thioureas according to example 1-53 givingexample 106-109 in total yields of 25-30%.

Examples 110-112

1H-imidazole-1-propanamine was reacted with the corresponding2-chlorobenzo[d]thiazole in toluol for 24 h at a temperature of 130° C.After removing the solvent and recristallization from methanol example110-112 was yielded in an amount of 55-65%.

Examples 113-118, 120-124 and 126-132

1H-imidazole-1-propanamine was reacted with the corresponding 2-phenylacetic acid in dry dioxane by adding one equivalent of CAIBE andN-methylmorpholine at a temperature of 0° C. After 2 h the mixture wasallowed to warm to r.t. and the mixture was stirred for 12 h. Afterremoving the solvent the resulting oil was redissolved in methylenechloride and the organic layer was washed by means of an aqueoussolution of NaHCO₃ and water, dried and the solvent was evaporated. Theremaining oil was dissolved in dioxane adding Laweson's Reagent. Afterstirring for 12 h a saturated solution of NaHCO₃ was added. Dioxane wasevaporated and the aqueous layer was extracted by means of ethylacetate. The organic layer was separated, dried and the solvent wasevaporated. The remaining solid was crystallized from acetylacetate/ether, giving 113-118, 120-124 and 126-132 with total yields of62-85%.

Example 119N-(3-(1H-imidazol-1-yl)propyl)-2-(3,4-dimethoxyphenyl)ethanethioamide

A mixture of 4.0 mmol triethylamine and 4.0 mmol of3-(1H-imidazol-1-yl)alkyl-1-amine mL of dioxane was added drop wise toan ice cooled, stirred solution of 4.0 mmol of2-(3,4-dimethoxyphenyl)acetyl chloride in 30 mL of dioxane. The mixturewas allowed to warm to r.t., and then stirred for 1 h. After removingthe solvent by reduced pressure, the residue was redissolved in 50 mL ofdichloromethane. The organic layer was washed by means of 30 mL ofsaturated aqueous solution of NaHCO₃, and water. The organic solutionwas dried, filtered, and the solvent was removed under reduced pressure.After redissolving in 50 mL of dry dioxane 2.2 mmol of Lawesson'sreagent was added, and the mixture was heated to 90° C. and stirred for8 h. The solvent was removed by reduced pressure, and the residue wasredissolved in 50 mL of dichloromethane. The organic layer was washedthree times by means of a saturated aqueous solution of NaHCO₃, followedthree times by water, dried, filtered, and then the organic solvent wasremoved. The compound was purified by chromatography using acentrifugal-force-chromatography device, (Harrison Research Ltd.)utilizing silica plates of a layer thickness of 2 mm, and a CHCl₃/MeOHgradient as eluting system.

Yield: 0.14 g (10.6%); melting point: 148.0-150.0° C.

¹H NMR δ 2.0-2.15 (br m, 2H), 3.4-3.5 (m, 2H), 3.7 (s, 6H), 6.75-6.8 (m,2H), 4.1-4.2 (m, 2H), 6.8-6.9 (m, 2H), 6.95-7.0 (m, 1H), 7.4 (s, 1H),7.75-7.85 (br m, 1H), 8.6 (s, 1H), 10.2 (s, 1H); MS m/z 320.2 (M+H),252.2 (M-C₃H₃N₂.)

Example 125N-(3-(1H-imidazol-1-yl)propyl)-1-(3,4-dimethoxyphenyl)cyclopropanecarbothioamide

11.06 mmol of 3,4-dimethoxyphenyl acetonitrile, 34.8 mmol of2-Bromo-1-chloroethanole and 1.16 mmol of triethylbenzylammoniumhydrochloride were dissolved in 10 mL of an aqueous solution of KOH(60%). The mixture was transferred into an ultrasonic bath andvigorously stirred for 3 h at room temperature. The resulting suspensionwas diluted with 40 mL of water and extracted three times by means of 20mL of dichloromethane. The combined organic layers where washed by meansof an aqueous solution of hydrochloric acid (1N), dried over Na₂SO₄ andthe solvent was removed under reduced pressure. The remaining oil waspurified by flash-chromatography using silica gel and ethylacetate/heptane as eluting system, resulting in 0.81 g (34.4%) of1-(3,4-dimethoxyphenyl)cyclopropanecarbonitrile

3.9 mmol of 1-(3,4-dimethoxyphenyl)cyclopropanecarbonitrile and 11.2mmol of KOH were suspended in 80 mL of ethylene glycol. The mixture wasstirred for 12 h under reflux. Then 80 mL of water were added and theaqueous layer was extracted two times with ether. After pH adjustment toa value of pH=4-5 using HCl (1N) the aqueous layer was extracted threetimes by means of ether, then the combined organic layers were driedover Na₂SO₄ and the solvent was removed, resulting in 0.81 g (93.5%) of1-(3,4-dimethoxyphenyl)cyclopropanecarboxylic acid.

3.44 mmol of 1-(3,4-dimethoxyphenyl)cyclopropanecarboxylic acid, 3.5mmol of N-Methyl morpholine, and 3.5 mmol of isobutyl chloroformiat weredissolved in dry tetrahydrofurane and stirred for 15 min at −15° C. Then3.5 mmol of 3-(1H-imidazol-1-yl)alkyl-1-amine was added and the mixturewas allowed to warm to 0° C. and was stirred for 12 h. The solvent wasremoved under reduced pressure and the remaining oil was redissolved inchloroform. Then the organic layer was washed two times by means of asaturated aqueous solution of NaHCO₃, then dried over Na₂SO₄ and thesolvent was removed. Purification was performed by means of centrifugalforced chromatography using a Chromatotron® device (Harrison ResearchLtd.) utilizing silica plates of a layer thickness of 2 mm, and aCHCl₃/MeOH gradient as eluting system resulting in 0.671 g (59.3%) ofN-(3-(1H-imidazol-1-yl)propyl)-1-(3,4-dimethoxyphenyl)cyclopropane-carboxamide.

After redissolving in 30 mL of dry dioxane 1.43 mmol of Lawesson'sreagent were added, and the mixture was heated to 90° C. and stirred for8 h. The solvent was removed by reduced pressure, and the residue wasremains were dissolved in 50 mL of dichloromethane. The organic layerwas washed three times by means of a saturated aqueous solution ofNaHCO₃, followed three times by water, dried, filtered, and then theorganic solvent was removed. The compound was purified by chromatographyusing a centrifugal-force-chromatography device, (Harrison ResearchLtd.) utilizing silica plates of a layer thickness of 2 mm, and aCHCl₃/MeOH gradient as eluting system.

Yield: 0.33 g (46.2%); melting point: 127.0-127.5° C.

¹H NMR δ 1.1-1.2 (t, 2H), 1.55-1.6 (t, 2H), 2.0-2.1 (m, 2H), 3.5-3.6 (m,2H), 3.7-3.8 (s, 6H), 4.1-4.2 (t, 2H), 6.8-6.9 (m, 3H), 7.65 (s, 1H),7.75 (s, 1H), 8.8 (m, 1H), 9.05 (s, 1H; MS m/z 346.0 (M+H), 278.2(M-C₃H₃N₂.), 177.1 (M-C₆H₈N₃S.)

Examples 133-135

A mixture of 1 equivalent triethylamine and 3,4-dimethoxyaniline indioxane was added to an stirred solution of the correspondingω-bromoalkyl acidic chloride at a temperature of 0° C. The solution wasallowed to warm to r.t. and stirred for 2 h. The solvent was evaporated,and the remaining oil was redissolved in dichloromethane. The organiclayer was washed by means of water, dried, filtered, and the solvent wasremoved under reduced pressure.

Imidazole and sodium hydride were suspended in and the mixture wasstirred under inert conditions at r.t. for 3 h.ω-Bromo-N-(3,4-dimethoxy-phenyl)alkylamide was added and the mixture washeated to 100° C. and stirred for 8 h. After that, the solvent wasevaporated, hot toluene were added and the solution was filtered. Thenthe solvent was removed under reduced pressure. The transformation intothe thioamides was performed as described for example 113-132 by meansof Laweson's reagent, giving 133-135 in total yields of 13-20%.

The analytical data for further examples, which were synthesizedaccording to the general synthesis schemes described above, are asfollows:

Example 1 1-(3-(1H-imidazol-1-yl)propyl)-3-methylthiourea

melting point: 122-122.5° C.

¹H NMR δ 1.85-1.95 (m, 2H), 2.8 (s, 3H), 3.2-3.5 (br d, 2H), 3.8-3.9 (m,2H), 6.85 (d, 1H), 7.15 (d, 1H), 7.3-7.5 (br d, 2H), 7.65 (s, 1H); MSm/z 199.1 (M+H), 221.3 (M+Na), 131.0 (M-C₃H₃N₂.)

Example 2 1-(3-(1H-imidazol-1-yl)propyl)-3-tert-butylthiourea

melting point: 147.0-147.5° C.

¹H NMR δ 1.3-1.4 (s, 9H), 1.85-1.95 (m, 2H), 3.5 (t, 2H), 3.8 (t, 2H),6.85 (d, 1H), 7.15 (d, 1H), 7.3-7.5 (br d, 2H), 7.65 (s, 1H); MS m/z241.1 (M+H), 173.1 (M-C₃H₃N₂.)

Example 3 1-(3-(1H-imidazol-1-yl)propyl)-3-benzylthiourea

melting point: 127.0-128.0° C.

¹H NMR δ 1.85-1.95 (m, 2H), 3.2-3.5 (br d, 2H), 3.8-3.9 (m, 2H), 4.6 (s,2H), 6.8 (d, 1H), 7.15 (d, 1H), 7.19-7.35 (m, 5H), 7.5-7.6 (br d, 2H),7.85 (s, 1H); MS m/z 275.3 (M+H), 207.1 (M-C₃H₃N₂.)

Example 5 1-(3-(1H-imidazol-1-yl)propyl)-3-phenylthiourea

melting point: 166.5-167.0° C.

¹H NMR δ 1.95-2.05 (m, 2H), 3.3-3.5 (br d, 2H), 3.9-4.0 (m, 2H), 6.85(d, 1H), 7.05 (m, 1H) 7.15 (d, 1H), 7.25 (m, 2H), 7.35 (m, 2H), 7.6 (s,1H), 7.8 (br s, 1H), 9.5 (br s, 1H); MS m/z 261.1 (M+H), 193.2(M-C₃H₃N₂.)

Example 6 1-(3-(1H-imidazol-1-yl)propyl)-3-(4-fluorophenyl)thiourea

melting point: 147.0-148.0° C.

¹H NMR δ 1.95-2.05 (m, 2H), 3.3-3.5 (br d, 2H), 3.9-4.05 (m, 2H), 6.85(d, 1H), 7.05-7.15 (m, 3H), 7.3-7.4 (m, 2H), 7.6 (s, 1H), 7.7-7.8 (br s,1H), 9.4 (br s, 1H); MS m/z 279.3 (M+H), 211.2 (M-C₃H₃N₂.)

Example 7 1-(3-(1H-imidazol-1-yl)propyl)-3-(4-ethylphenyl)thiourea

melting point: 100.0-100.5° C.

¹H NMR δ 1.15-1.2 (t, 3H), 1.9-2.0 (m, 2H), 2.5-2.6 (m, 2H), 3.3-3.5 (brd, 2H), 3.9-4.05 (m, 2H), 6.85 (d, 1H), 7.1-7.2 (m, 3H), 7.25-7.3 (m,2H), 7.6 (s, 1H), 7.7-7.8 (br s, 1H), 9.4 (br s, 1H); MS m/z 289.3(M+H), 221.1 (M-C₃H₃N₂.)

Example 81-(3-(1H-imidazol-1-yl)propyl)-3-(4-(trifluoromethyl)phenyl)thiourea

melting point: 154.5-155.0° C.

¹H NMR δ 1.9-2.1 (br m, 2H), 3.4-3.6 (br d, 2H), 3.95-4.1 (br m, 2H),6.85 (d, 1H), 7.2 (d, 1H), 7.6-7.8 (m, 5H), 8.2 (br s, 1H), 9.9 (br s,1H); MS m/z 329.3 (M+H), 261.2 (M-C₃H₃N₂.)

Example 10 1-(3-(1H-imidazol-1-yl)propyl)-3-(4-acetylphenyl)thiourea

melting point: 170.0-171.0° C.

¹H NMR δ 1.9-2.1 (br m, 2H), 2.4-2.5 (s, 3H), 3.2-3.5 (br m, 2H),3.9-4.1 (m, 2H), 6.85 (d, 1H), 7.15 (d, 1H), 7.5-7.65 (br m, 3H),7.8-7.9 (m, 2H), 8.1 (m, 2H), 9.8 (br s, 1H); MS m/z 303.2 (M+H), 235.1(M-C₃H₃N₂.)

Example 11 1-(3-(1H-imidazol-1-yl)propyl)-3-(4-methoxyphenyl)thiourea

melting point: 125.0-125.5° C.

¹H NMR δ 1.8-2.0 (br m, 2H), 3.2-3.5 (br m, 2H), 3.7 (s, 3H), 3.9-4.0(m, 2H), 6.7-6.9 (m, 3H), 7.1-7.2 (m, 3H), 7.5 (s, 1H), 7.6 (s, 1H), 9.2(s, 1H); MS m/z 291.1 (M+H), 223.2 (M-C₃H₃N₂.)

Example 141-(3-(1H-imidazol-1-yl)propyl)-3-(2,4-dimethoxyphenyl)thiourea

melting point: 120.0-120.5° C.

¹H NMR δ 1.8-2.0 (br m, 2H), 3.4-3.5 (br m, 2H), 3.75 (s, 6H), 3.9-4.0(m, 2H), 6.5 (d, 1H), 6.6 (s, 1H), 6.9 (s, 1H), 7.15 (s, 1H), 7.3 (d,1H), 7.5 (br s, 1H), 7.6 (s, 1H), 9.75 (s, 1H); MS m/z 321.2 (M+H),253.3 (M-C₃H₃N₂.)

Example 151-(3-(1H-imidazol-1-yl)propyl)-3-(3,5-dimethoxyphenyl)thiourea

melting point: 142.0-143.0° C.

¹H NMR δ 1.8-2.0 (br m, 2H), 3.4-3.5 (br m, 2H), 3.6 (s, 6H), 3.95-4.0(m, 2H), 6.25 (m, 1H), 6.6 (m, 2H), 6.9 (s, 1H), 7.2 (s, 1H), 7.6 (s,1H), 7.8 (s, 1H), 9.5 (s, 1H); MS m/z 321.2 (M+H), 253.3 (M-C₃H₃N₂.)

Example 231-(3-(1H-imidazol-1-yl)propyl)-3-(2,3-dihydrobenzo[b][1,4]dioxin-7-yl)-thiourea

melting point: 103.0-103.5° C.

¹H NMR δ 1.9-2.0 (br m, 2H), 3.3-3.5 (br d, 2H), 3.9-4.0 (m, 2H),4.2-4.3 (m, 4H), 6.7 (m, 1H), 6.8-6.8 (m, 1H), 6.9 (m, 2H), 7.2 (s, 1H),7.6 (m, 2H), 9.3 (s, 1H); MS m/z 319.3 (M+H), 251.3 (M-C₃H₃N₂.)

Example 241-(3-(1H-imidazol-1-yl)propyl)-3-(benzo[d][1,3]dioxol-6-yl)thiourea

melting point: 115.0-115.6° C.

¹H NMR δ 1.9-2.1 (br m, 2H), 3.4-3.5 (br d, 2H), 4.05-4.15 (m, 2H), 6.0(s, 2H), 6.7 (m, 1H), 6.8-6.85 (m, 1H), 6.95 (d, 1H), 7.25 (s, 1H), 7.45(s, 1H), 7.7 (br s, 1H), 8.5 (br s, 1H), 9.4 (br s, 1H); MS m/z 305.2(M+H), 237.2 (M-C₃H₃N₂.)

Example 251-(3-(1H-imidazol-1-yl)propyl)-3-(3,4,5-trimethoxyphenyl)thiourea

melting point: 124.5-125.5° C.

¹H NMR δ 1.8-2.0 (m, 2H), 3.4-3.5 (br m, 2H), 3.6 (s, 3H), 3.7 (s, 6H),3.9-4.0 (m, 2H), 6.65 (m, 2H), 6.85 (s, 1H), 7.2 (s, 1H), 7.6 (s, 1H),7.7 (br s, 1H), 9.4 (s, 1H); MS m/z 351.3 (M+H), 283.2 (M-C₃H₃N₂.)

Example 26 1-(3-(1H-imidazol-1-yl)propyl)-3-(3-methoxyphenyl)thiourea

melting point: 89.5-90.0° C.

¹H NMR δ 1.9-2.1 (br m, 2H), 3.4-3.5 (br m, 2H), 3.7 (s, 3H), 3.9-4.0(m, 2H), 6.6-6.7 (m, 1H), 6.8-6.9 (m, 2H), 7.1 (m, 2H), 7.15-7.25 (br m,1H), 7.6 (s, 1H), 7.8 (br s, 1H), 9.5 (s, 1H); MS m/z 291.1 (M+H), 223.2(M-C₃H₃N₂.)

Example 27 1-(3-(1H-imidazol-1-yl)propyl)-3-(4-ethoxyphenyl)thiourea

melting point: 126.0-126.5° C.

¹H NMR δ 1.5 (br m, 3H), 1.9-2.0 (br m, 2H), 3.4-3.5 (br m, 2H), 3.9-4.0(br m, 4H), 6.8-6.9 (m, 2H), 6.95 (s, 1H), 7.15-7.2 (m, 2H), 7.25 (s,1H), 7.55-7.6 (br s, 1H), 7.8 (s, 1H), 9.3 (s, 1H); MS m/z 305.2 (M+H),237.2 (M-C₃H₃N₂.)

Example 331-(3-(1H-imidazol-1-yl)propyl)-3-(4-(methylthio)phenyl)thiourea

melting point: 140.0-140.5° C.

¹H NMR δ 1.8-2.05 (br m, 2H), 2.5 (s, 3H), 3.3-3.5 (br m, 2H), 3.9-4.1(m, 2H), 6.9 (m, 1H), 7.1-7.3 (br m, 5H), 7.6 (s, 1H), 7.75 (br s, 1H),9.4 (s, 1H); MS m/z 307.2 (M+H), 239.2 (M-C₃H₃N₂.)

Example 42 1-(3-(1H-imidazol-1-yl)propyl)-3-(4-nitrophenyl)thiourea

melting point: 165.0. 166.0° C.

¹H NMR δ 1.9-2.05 (m, 2H), 3.3-3.5 (br d, 2H), 3.95-4.05 (m, 2H), 6.85(d, 1H), 7.15 (d, 1H), 7.6 (d, 1H), 7.7 (m, 2H), 8.1 (m, 2H), 8.3 (br s,1H), 10.1 (br s, 1H); MS m/z 306.2 (M+H), 237.9 (M-C₃H₃N₂.)

Example 501-(3-(1H-imidazol-1-yl)propyl)-3-(4-(dimethylamino)phenyl)thiourea

melting point: 146.5-147.0° C.

¹H NMR δ 1.9-2.0 (m, 2H), 2.9 (s, 6H), 3.4 (m, 2H), 3.9-4.0 (m, 2H), 6.7(m, 2H), 6.9 (s, 1H), 7.05-7.1 (m, 2H), 7.15 (s, 1H), 7.4 (br s, 1H),7.6 (s, 1H), 9.2 (s, 1H); MS m/z 304.2 (M+H), 236.0 (M-C₃H₃N₂.)

Example 102 1-(3-(1H-imidazol-1-yl)propyl)-3-(3,4-dimethoxyphenyl)urea

melting point: 114.5-115.0° C.

¹H NMR δ 1.7-1.9 (m, 2H), 2.9-3.1 (m, 2H), 3.7 (2s, 6H), 3.9-4.0 (m,2H), 6.1 (t, 1H), 6.7 (s, 2H), 6.8 (s, 1H), 7.15 (d, 2H), 7.6 (s, 1H),8.2 (s, 1H); MS m/z 321.2 (M+H), 253.3 (M-C₃H₃N₂.)

Example 1061-((S)-3-(1H-imidazol-1-yl)-2-methylpropyl)-3-(3,4-dimethoxyphenyl)-thiourea

melting point: 150.5-151.5° C.

¹H NMR δ 0.9 (d, 3H), 2.3-2.4 (m, 2H), 2.5 (s, 1H), 3.7 (d, 6H), 4.0-4.1(br m, 1H), 4.15-4.25 (br m, 1H), 6.75-6.8 (m, 1H), 6.85 (m, 1H),6.9-7.0 (m, 1H), 7.65 (s, 1H), 7.75 (s, 2H), 9.1 (s, 1H), 9.5 (s, 1H);MS m/z 335.6 (M+H), 267.1 (M-C₃H₃N₂.)

Example 1071-((R)-3-(1H-imidazol-1-yl)-2-methylpropyl)-3-(3,4-dimethoxyphenyl)-thiourea

melting point: 155.0-157.5° C.

¹H NMR δ 0.9 (d, 3H), 2.3-2.4 (m, 2H), 2.5 (s, 1H), 3.7 (d, 6H), 4.0-4.1(br m, 1H), 4.15-4.25 (br m, 1H), 6.75-6.8 (m, 1H), 6.85 (m, 1H),6.9-7.0 (m, 1H), 7.65 (s, 1H), 7.75 (s, 2H), 9.1 (s, 1H), 9.5 (s, 1H);MS m/z 335.4 (M+H), 267.2 (M-C₃H₃N₂.)

Example 1091-((1-((1H-imidazol-1-yl)methyl)cyclopropyl)methyl)-3-(3,4-dimethoxy-phenyl)thiourea

melting point: 166.5-168.5° C.

¹H NMR δ 0.7-0.8 (br m, 2H), 1.85-1.9 (m, 1H), 2.15-2.2 (m, 1H), 2.2-2.3(m, 1H), 3.4-3.5 (m, 1H), 3.7 (d, 6H), 4.2 (s, 1H), 4.95 (s, 1H),6.75-6.8 (br m, 1H), 6.85-6.9 (br m, 1H), 7.0 (s, 1H), 7.5 (m, 1H), 7.6(m, 1H), 7.7 (s, 0.5H), 7.8 (s, 0.5H), 8.85 (s, 0.5H), 9.1 (s, 0.5H),9.35 (s, 0.5H), 9.45 (s, 0.5H); MS m/z 347.2 (M+H), 279.2 (M-C₃H₃N₂.),137.5 (M-C₉H₁₃N₄S.)

Example 110 N-(3-(1H-imidazol-1-yl)propyl)benzo[d]thiazol-2-amine

¹H NMR δ 1.95-2.15 (m, 2H), 3.25-3.35 (m, 2H), 4.0-4.1 (t, 2H), 6.9 (s,1H), 6.95-7.05 (t, 1H), 7.15-7.2 (m, 2H), 7.35-7.4 (d, 1H), 7.60-7.70(m, 2H), 8.0-8.1 (br s, 1H); MS m/z 259.4 (M+H), 191.3 (M-C₃H₃N₂.)

Example 111N-(3-(1H-imidazol-1-yl)propyl)-6-chlorobenzo[d]thiazol-2-amine

¹H NMR δ 1.95-2.15 (m, 2H), 3.25-3.35 (m, 2H), 4.0-4.1 (t, 2H), 6.9 (s,1H), 7.1-7.2 (d, 2H), 7.3-7.4 (d, 1H), 7.65 (s, 1H), 7.8 (s, 1H), 8.2(s, 1H); MS m/z 293.3 (M+H), 225.3 (M-C₃H₃N₂.)

Example 112N-(3-(1H-imidazol-1-yl)propyl)-6-methoxybenzo[d]thiazol-2-amine

¹H NMR δ 1.9-2.05 (m, 2H), 3.2-3.3 (m, 2H), 3.7 (s, 3H), 4.0-4.1 (t,2H), 6.7-6.8 (d, 1H), 6.9 (s, 1H), 7.15-7.2 (s, 1H), 7.2-7.3 (m, 2H),7.65 (s, 1H), 7.8 (s, 1H); MS m/z 289.1 (M+H), 221.4 (M-C₃H₃N₂.)

Example 115 (R)-N-(3-(1H-imidazol-1-yl)propyl)-2-phenylpropanethioamide

melting point: 82.0-82.5° C.

¹H NMR δ 1.4-1.55 (d, 3H), 1.9-2.0 (m, 2H), 3.4-3.5 (m, 2H), 3.85-3.95(m, 2H), 4.0-4.1 (q, 1H), 6.8-6.9 (s, 1H), 7.1 (s, 1H), 7.15-7.2 (m,1H), 7.2-7.3 (m, 2H), 7.35-7.4 (m, 2H), 7.55 (s, 1H), 10.1 (s, 1H); MSm/z 274.4 (M+H), 206.3 (M-C₃H₃N₂.)

Example 116 (S)-N-(3-(1H-imidazol-1-yl)propyl)-2-phenylpropanethioamide

melting point: 82.5-83.5° C.

¹H NMR δ 1.4-1.55 (d, 3H), 1.9-2.0 (m, 2H), 3.4-3.5 (m, 2H), 3.85-3.95(m, 2H), 4.0-4.1 (q, 1H), 6.8-6.9 (s, 1H), 7.1 (s, 1H), 7.15-7.2 (m,1H), 7.2-7.3 (m, 2H), 7.35-7.4 (m, 2H), 7.55 (s, 1H), 10.1 (s, 1H); MSm/z 274.4 (M+H), 206.3 (M-C₃H₃N₂.)

Example 121N-(3-(1H-imidazol-1-yl)propyl)-1-(4-chlorophenyl)cyclobutanecarbo-thioamide

melting point: 137.5-139.0° C.

¹H NMR δ 1.55-1.75 (br m, 2H), 1.85-1.95 (br m, 2H), 2.4-2.5 (br m, 2H),2.7-2.85 (br m, 2H), 3.3-3.5 (br m, 2H), 3.8 (m, 2H), 6.9 (s, 1H), 7.0(s, 1H), 7.3 (m, 2H), 7.45 (s, 1H), 7.5 (m, 2H), 9.6 (t, 1H); MS m/z334.3 (M+H), 266.1 (M-C₃H₃N₂.)

Example 122N-(3-(1H-imidazol-1-yl)propyl)-1-(4-chlorophenyl)cyclopentanecarbo-thioamide

melting point: 140.0-141.0° C.

¹H NMR δ 1.5-1.65 (br m, 4H), 1.8-1.9 (m, 2H), 2.0-2.1 (m, 2H), 2.6 (m,2H), 3.4-3.5 (m, 2H), 3.7-3.8 (m, 2H), 6.85 (s, 1H), 7.0 (s, 1H), 7.35(m, 2H), 7.4 (m, 2H), 7.5 (s, 1H), 9.4 (t, 1H); MS m/z 348.2 (M+H),280.2 (M-C₃H₃N₂.)

Example 123N-(3-(1H-imidazol-1-yl)propyl)-1-(4-methoxyphenyl)cyclohexanecarbo-thioamide

melting point: 162.5-164.0° C.

¹H NMR δ 1.2-1.3 (m, 1H), 1.35-1.5 (br m, 5H), 1.85-2.0 (br m, 4H),2.4-2.6 (br m, 2H), 3.4-3.5 (m, 2H), 3.7 (s, 3H), 3.8 (m, 2H), 6.8 (m,3H), 7.0 (s, 1H), 7.3 (m, 2H), 7.5 (s, 1H), 9.2 (t, 1H); MS m/z 358.3(M+H), 290.3 (M-C₃H₃N₂.)

Example 124N-(3-(1H-imidazol-1-yl)propyl)-1-(4-methoxyphenyl)cyclopropanecar-bothioamide

melting point: 129.0-129.5° C.

¹H NMR δ 1.0-1.1 (m, 2H), 1.5-1.6 (m, 2H), 1.9-2.0 (br m, 2H), 3.4-3.5(m, 2H), 3.7 (s, 3H), 3.9 (m, 2H), 6.9 (m, 3H), 7.1 (s, 1H), 7.2-7.3 (m,2H), 7.6 (s, 1H), 8.9 (br s, 1H); MS m/z 316.0 (M+H), 248.4 (M-C₃H₃N₂.)

Example 134 5-(1H-imidazol-1-yl)-N-(3,4-dimethoxyphenyl)pentanethioamidemelting point: 128.0-128.5° C.

¹H NMR δ 1.65-1.70 (m, 2H), 1.75-1.80 (m, 2H), 2.7-2.75 (m, 2H), 3.7 (s,3H), 3.75 (s, 3H), 4.0-4.05 (t, 2H), 6.9-7.0 (m, 2H), 7.2 (s, 1H), 7.3(d, 1H), 7.5 (s, 1H), 7.75 (s, 1H), 11.0 (s, 1H); MS m/z 320.2 (M+H),252.2 (M-C₃H₃N₂.)

Example 1361-(2-(1H-imidazol-1-yl)ethyl)-3-(3,4-dimethoxyphenyl)thiourea

melting point: 157.5-159.0° C.

¹H NMR δ 3.7 (2 s, 6H), 3.8 (m, 2H), 4.2 (m, 2H), 6.7 (m, 1H), 6.85 (m,1H), 6.9 (m, 2H), 7.15 (s, 1H), 7.5 (br s, 1H), 7.6 (s, 1H), 9.5 (s,1H); MS m/z 307.2 (M+H), 239.1 (M-C₃H₃N₂.)

ABBREVIATIONS

° C. degree CelsiusA, Ala alanine

Aβ amyloid-β peptide

ABri amyloid peptide in familial british dementiaAC adenylyl cyclaseADan amyloid peptide in familial danish dementiaAIM absent in melanomaAMC amino methyl coumarineas antisenseAsp aspartateβNA beta-naphtylamineBA butyric acidbp basepairBSA bovine serum albuminC cysteineCAT chloramphenicol acetyl transferasecAMP cyclic adenosine monophsphateCCL2 MCP-1, monocyte chemoattractant protein 1CCL7 MCP-3, monocyte chemoattractant protein 3CCL8 MCP-2, monocyte chemoattractant protein 2CCL13 MCP-4, monocyte chemoattractant protein 4cDNA copy-DNAC-His C-terminal histidine tagCIDP Chronic inflammatory demyelinizing polyradiculoneuropathyCl chlorineCSF cerebor-spinal fluid (liquor cerebrospinalis)C-terminus carboxy-terminusCTL cytotoxic T-lymphocyteCV column volumed diameter

Da Dalton

DMSO dimethyl sulphoxideDNA desoxyribonucleic acidE enzymeEBV Epstein Barr virusECL enterochromaffin-likeE. coli Escherichia coliEC glutamyl cyclaseED effective doseEGFP enhanced green fluorescent proteinES enzyme-substrate complexFPP fertilization promoting peptideFTC follicular thyroid carcinomag relative centrifugal forceGBS Guillain-Barré syndromeGF gel filtrationGln glutamineGlu glutamic acidGnRH gonadotropin-releasing hormone (gonadoliberin)GST glutathion S-transferaseH hydrogenh human, hourHGF hepatocyte growth factorHIC hydrophobic interaction chromatographyHIF1a hypoxia induced factor 1aHis histidineHPLC high performance liquid chromatographyI inhibitor, isoleucineID identificationIMAC immobilized metal affinity chromatography

IPTG Isopropyl-β-D-thiogalactopyranosid

K potassiumk constantkDA kilo-daltonk_(i) inhibitor constantKLH Keyhole limpet hemocyaninI length

LB Luria-Bertani

LD lethal doseLPS lipopolysaccharideM molarμl micro-literμM micro-molarMAGEA melanoma antigen family AMAGEB melanoma antigen family BMaldi-tof matrix assisted laser desorption/ionization time-of-flightMART 1 melanoma antigen recognized by T-cells 1max maximumMCL-1 myeloid cell leukemia 1Met methioninemin minutesmM milli-molar

MS Multiple Sclerosis

mRNA messenger-RNAN asparagineNa sodiumNADH nicotinamide adenine dinucleotidenm nanometerNO number

NT Neurotensin

N-terminus amino terminusO oxygenOD optical densityP product, phosphorPBS phosphate-buffered salinePCR polymerase chain reactionpGlu pyroglutamic acidpH pondus hydrogeniiPro prolinePTC papillary thyroid carcinomaPyr pyroglutamateQC glutaminyl cyclase (glutaminyl-peptide cyclotransferase)qPCR quantitative real-time polymerase chain reactionQPCTL glutaminyl-peptide cyclotransferase—likeRNA ribonucleic acidRT reverse transcription; reverse transcriptaseS substrates senseSAGE serial analysis of gene expressionSDS sodium dodecly sulfateSDS-PAGE SDS-polyacrylamid gelelectrophoresisSGAP Streptomyces griseus amino peptidaseSEQ sequenceSNP single nucleotide polymorphismtaa tumor-associated antigenTGF-β transforming growth factor betaTNF-α tumor necrosis factor alphaTRH thyreotropin-realeasing hormone (thyreoliberin)TSH thyroidea-stimulating-hormoneTYR tyrosinaseTYRP tyrosinase related proteinU unitUTC undifferentiated thyroid carcinomaUV ultravioletV velocityVpAP Vibrio proteolytica amino peptidaseYSS yeast signal sequenceZn zinc

1. A method of screening for a compound capable of inhibiting anenzymatic activity, comprising: incubating a modified cell thatexpresses at least a first polypeptide and a suitable substrate for thefirst polypeptide in the presence of at least one test compound or saltthereof; measuring an enzymatic activity of the first polypeptide;comparing said activity with enzymatic activity determined in theabsence of the at least one test compound or salt thereof; and selectinga test compound that reduces the enzymatic activity of the firstpolypeptide; wherein the first polypeptide comprises: (a) an amino acidsequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ IDNO: 17, SEQ ID NO: 18, SEQ ID NO: 21, and SEQ ID NO: 22, or an aminoacid sequence having at least about 95% sequence identity thereto andhaving glutaminyl cyclase activity; (b) an amino acid sequence encodedby a nucleic acid sequence selected from the group consisting of SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 19, and SEQ ID NO: 20; or(c) a fragment of (a) or (b) wherein said fragment is immunologicallyreactive and has glutaminyl cyclase activity.
 2. The method of claim 1,wherein the first polypeptide comprises an amino acid sequence having atleast about 95% sequence identity to an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ IDNO: 18, SEQ ID NO: 21, or SEQ ID NO: 22 and has glutaminyl cyclaseactivity.
 3. The method of claim 1 wherein the enzymatic activity isglutaminyl cyclase activity.
 4. The method of claim 3 furthercomprising: incubating a modified cell that expresses a secondpolypeptide comprising SEQ ID NO: 10 (wild type human glutaminylcyclase) and a suitable substrate for wild type human glutaminyl cyclasein the presence of the at least one test compound or salt thereof;measuring the glutaminyl cyclase activity of the second polypeptide; andselecting a test compound that reduces the glutaminyl cyclase activityof the first first polypeptide but does not reduce the glutaminylcyclase activity of the second polypeptide.
 5. The method of claim 1wherein the first polypeptide comprises: the amino acid sequence of SEQID NO: 21, or an amino acid sequence having at least about 95% sequenceidentity to SEQ ID NO: 21 and having glutaminyl cyclase activity; theamino acid sequence encoded by a nucleic acid sequence of SEQ ID NO: 19,or at least about 95% sequence identity to SEQ ID NO: 19 and havingglutaminyl cyclase activity; or a fragment thereof wherein said fragmentis immunologically reactive and has glutaminyl cyclase activity.
 6. Themethod of claim 1 wherein the first polypeptide comprises: the aminoacid sequence of SEQ ID NO: 21; the amino acid sequence having at leastabout 95% sequence identity to SEQ ID NO: 21 and having glutaminylcyclase activity; or a fragment thereof wherein said fragment isimmunologically reactive and has glutaminyl cyclase activity.
 7. Themethod of claim 6 wherein the first polypeptide comprises a fragment ofSEQ ID NO: 21, said fragment comprises amino acid residues 11 through392 of SEQ ID NO: 21, and said fragment is immunologically reactive andhas glutaminyl cyclase activity; or an amino acid sequence having atleast about 95% sequence identity to said fragment and having glutaminylcyclase activity.
 8. The method of claim 1 wherein the first polypeptidecomprises: the amino acid sequence of SEQ ID NO: 21; the amino acidsequence having at least about 99% sequence identity to SEQ ID NO: 21and having glutaminyl cyclase activity; or a fragment thereof whereinsaid fragment is immunologically reactive and has glutaminyl cyclaseactivity.
 9. The method of claim 1 wherein the first polypeptide isglycosylated.
 10. The method of claim 1 wherein the first polypeptide isfree in solution; affixed to a solid support; borne on a cell surface;or located intracellularly.
 11. The method of claim 1 wherein measuringthe enzymatic activity of the first polypeptide comprises measuringglutaminyl cyclase activity of the first polypeptide.
 12. The method ofclaim 1 wherein selecting a test compound that reduces the enzymaticactivity of the first polypeptide comprises selecting a test compoundwith a Ki for glutaminyl cyclase activity inhibition of 10 μM or less.13. The method of claim 1 wherein selecting a test compound that reducesthe enzymatic activity of the first polypeptide comprises selecting atest compound with a Ki for glutaminyl cyclase activity inhibition of 1μM or less.
 14. The method of claim 1 wherein selecting a test compoundthat reduces the enzymatic activity of the first polypeptide comprisesselecting a test compound with a Ki for glutaminyl cyclase activityinhibition of 0.1 μM or less.
 15. The method of claim 1 whereinselecting a test compound that reduces the enzymatic activity of thefirst polypeptide comprises selecting a test compound with a Ki forglutaminyl cyclase activity inhibition of 0.01 μM or less.
 16. Themethod of claim 1, wherein the suitable substrate comprises Glu¹-A Bri(SEQ ID NO: 33), Glu¹-ADan (SEQ ID NO: 34), Gln¹-Gastrin 17 (SEQ ID NO:35), Gln¹-Gastrin 34 (SEQ ID NO: 36), Gln¹-neurotensin (SEQ ID NO: 41),Gln¹-fertilization promoting peptide (FPP) (QEP amino acid sequence),Gln¹-thyrotrophin releasing hormone (TRH) (QHP amino acid sequence),Gln¹-CCL2 (SEQ ID NO: 45), Gln¹-CCL7 (SEQ ID NO: 48), Gln¹-CCL8 (SEQ IDNO: 44), Gln¹-CCL16 (SEQ ID NO: 43), Gln¹-CCL18 (SEQ ID NO: 46),Gln¹-fractalkine (SEQ ID NO: 47), Gln¹-orexin A (SEQ ID NO: 49) orpeptide QYNAD (SEQ ID NO: 51).
 17. The method of claim 1, whereinmeasuring an enzymatic activity of the first polypeptide comprises afluorometric assay.
 18. The method of claim 1, wherein measuring anenzymatic activity of the first polypeptide comprises a massspectrophotometric assay.
 19. The method of claim 1, wherein measuringan enzymatic activity of the first polypeptide occurs at a pH of 7 to 8.